CN109942674B - GPR1 antagonistic polypeptide, and derivative and application thereof - Google Patents

Abstract

The invention discloses a GPR1 antagonistic polypeptide and a derivative and application thereof, and particularly relates to a sequence SEQ ID No.1-7 and a derivative thereof, wherein the derivative of a binding peptide is a product obtained by carrying out conventional modification on a GPR1 binding peptide amino acid side chain group and an amino end or a carboxyl end of a GPR1 antagonistic polypeptide fragment, or a product obtained by connecting a label for polypeptide or protein detection or purification to a GPR1 antagonistic polypeptide; the binding peptide and its derivatives can bind to GPR1 in vitro, promote cAMP concentration increase by blocking binding of chemerin and GPR1, and inhibit calcium (Ca) caused by chemerin²⁺) The internal flow provides an effective small molecule medicament for treating diseases of high expression GPR1 receptor, such as breast cancer, and the like, and can be widely applied to the fields of medicine and biology.

Description

GPR1 antagonistic polypeptide, and derivative and application thereof

Technical Field

The invention relates to the field of biotechnology and biomedicine, in particular to female reproductive disease target GPR1 receptor antagonist polypeptide LRH7-G2 and derivatives and application thereof.

Background

Breast cancer (Breast cancer) is a worldwide 2 nd highest mortality fatal cancer next to lung cancer that continues to undermine the life health of millions of women and their families worldwide. According to the latest statistics of the world health organization international cancer research center, the number of new breast cancer cases of women in 2012 worldwide reaches 167 thousands, and accounts for 22.9 percent of the incidence of all female malignant tumors; 46 ten thousand women died due to breast cancer, accounting for 13.7% of all women's malignant tumor deaths, accounting for 1.7% of all women's deaths, and about 1 out of 4 women with cancer had breast cancer. The age-normalized incidence rate of breast cancer of Chinese women is 21.6/10 ten thousand, and the Chinese women are in the 1 st position of the incidence rate of the breast cancer; the mortality rate is 5.7/10 ten thousand, and the number 6 of cancer deaths of women is located. 4 to 6 percent of breast cancer is metastatic breast cancer when being diagnosed, 30 to 40 percent of early patients receiving adjuvant therapy can develop metastatic breast cancer, and the 5-year survival rate of the patients is about 20 percent. However, the traditional treatment modes such as surgery, radiotherapy and chemotherapy have poor specificity and specificity of action, inevitably generate killing effect on normal cells and tissues and bring great side effect to patients. Therefore, the search for highly effective and specific target drugs will greatly improve the treatment effect of breast cancer!

Clinical researches find that early-stage breast cancer does not have typical symptoms and signs, is not easy to attach importance, and can be found only by physical examination or breast cancer screening. Although the early diagnosis of the breast cancer can win precious time for the treatment of the breast cancer and can greatly improve the treatment effect of the breast cancer, the proportion of people for carrying out the early diagnosis of the breast cancer in China is very small at present.

Metastasis to lymph after breast cancer surgery is a common metastatic symptom of breast cancer. Generally, lateral breast disease has a chance of metastasizing to axillary lymph nodes of 2/3 and parasternal metastasis of 1/3. Tumors in the medial and central regions of the breast have 1/2 chance of lymphatic metastasis to medial and lateral breast cancer, and lung metastasis and lymphatic metastasis are likely to occur in breast cancer. The tumor microenvironment is crucial to the formation of tumors, which determines the occurrence and development of tumors. Firstly, normal breast stem cells are converted into breast cancer stem cells through oncogenic mutation to generate tumors with poor prognosis, and then a part of the breast cancer stem cells with poor prognosis are transferred to the brain, the lung, the liver and the bone marrow under the influence of stromal fibroblasts of the tumors with poor prognosis and metastatic property.

Research shows that the chemokine receptor CXCR4 is abnormally and highly expressed in breast cancer tissues, the ligand stromal cell derived factor-1 (CXCL12) is also highly expressed in metastatic parts of breast cancer, such as bone marrow, lymph nodes, lung and liver, and a CXCR4/CXCL12 signal system plays an important role in the homing of the breast cancer to the metastasis of other distant organs, so that the CXCR4 antagonistic polypeptide CTCE-9908 also becomes a potential therapeutic target of the bone metastasis of the breast cancer, enters a preclinical test stage, and can well inhibit the bone metastasis of the breast cancer in a breast cancer animal model. Therefore, the chemotactic factor plays an important role in the occurrence, development and metastasis of the breast cancer, and the targeted drug for treating the breast cancer by taking the chemotactic factor as a target has wide market prospect and application value.

Breast cancer originates from the ductal and acinar epithelia at various levels of the breast, and develops gradually from hyperplasia to atypical hyperplasia of the glandular epithelium into carcinoma in situ, early invasive carcinoma to invasive carcinoma. The tissue types often differ from one grade of catheter to another. More than 95% of breast cancers are malignant epithelial tumors, and mammary sarcoma is very rare. The two most common histopathologically invasive breast cancers are: invasive ductal carcinoma (also known as "general type, NOS") and lobular carcinoma. Both breast cancers are formed by pre-invasive ductal and lobular neoplasias. Invasive ductal carcinoma and lobular carcinoma, as well as other types of breast cancer, differ primarily by morphological differences between cells.

The breast cancer molecule targeted therapy refers to therapy aiming at signal pathways related to the occurrence and development of breast cancer and related expression products of oncogenes of the breast cancer. Under the influence of high incidence of malignant tumors, the global market for antitumor drugs shows a continuous increase, the sales of the antitumor drugs in 2010 reaches $ 459.7 billion, which accounts for 5.8% of the sales of the drugs in the world, and the worldwide first, the global market is 8% higher than that in 2009. Wherein, the sales of the molecular targeting antitumor drug is $ 311.9 hundred million, and the growth rate reaches 23 percent. In 1997, the U.S. Food and Drug Administration (FDA) approved the use of a trastuzumab monoclonal antibody, a molecular targeted therapeutic against human epidermal growth factor receptor 2(HER-2), and began a new era in molecular targeted therapy of breast cancer. According to the statistics of global marketable drug data, the global breast cancer drug market size in 2015 exceeds $ 100 billion, wherein the highest sales is Trastuzumab (Trastuzumab), the sales is $ 67.9 billion, and the increase is 4.2% compared with the same period. Many studies have found that Epidermal Growth Factor Receptor (EGFR) is a key molecule in regulating the development and progression of breast cancer, and that EGFR promotes proliferation, migration, and invasion of breast cancer by activating downstream Ras/Raf/MEK/ERK and PI3K/AKT signaling pathways; in addition, epidermal growth factor receptor 2(HER-2) is highly expressed in breast cancer cells, and activation of downstream PI3K/Akt and Ras/MAPK signaling pathways promotes breast cancer proliferation, angiogenesis, migration and invasion by forming homodimers or heterodimers. At present, the molecular targeted drugs for treating breast cancer mainly comprise: trastuzumab for epidermal growth factor receptor-2 therapy; everolimus, an inhibitor in the PI3K/AKT/mTOR pathway; bevacizumab against anti-angiogenic drugs; iniparib directed against the BRCA1/2 mutation; and against the CDK4/6 inhibitor palbociclib. The advent and application of these targeted drugs brought new eosin to breast cancer patients, which made the treatment of breast cancer more accurate and greatly reduced the side effects of injury from traditional treatments!

In the development process of breast cancer, the chemotactic factor and the receptor thereof are closely related to the establishment of a tumor microenvironment and tumor metastasis, and can mediate the deterioration of tumor cells and promote the growth and proliferation of the tumor cells. Research indicates that chemokines such as CXCL8, CXCL12, CCL2, CCL5, CCL18, and the like have a breast cancer promoting effect, and other chemokines such as XCL1, CXCL9, CXCL10, CXCL14, CCL16, CCL19, and the like may have an anti-breast cancer effect. Tumor-associated fibroblasts (CAFs) and cancer cells can activate and secrete a large amount of CCL2, promote self-renewal of tumor stem cells (CSC), and further strengthen the resistance of tumor cells to chemo-radiotherapy and promote metastasis of lesions. The positive rate of CXCR4 in breast cancer is up to 60%, the CXCL12/CXCR4 action network plays an important role in the growth and metastasis of various solid tumors including breast cancer, and the blocking of the biological axis can inhibit angiogenesis and tumor spread and increase the sensitivity of tumor cells to chemotherapy and radiotherapy. In the 4T1 mouse breast cancer model, treatment with oncolytic viruses carrying CXCR4 antagonists can inhibit angiogenesis and tumor metastasis. The expression (71%) of CXCR4 in TNBC is higher than that of HER-2 positive breast cancer (44%) and Luminal type breast cancer (37%), and CXCR4+ TNBC patients have large tumors, are easy to generate liver, lung and brain metastasis and have poor prognosis. The chemotactic factor and the receptor antagonist or inhibitor thereof have wide market prospect. Statistics show that about 30% of small molecule drugs are targeted to G protein-coupled receptors. The CXCR4 polypeptide antagonist CTCE-9908 has been shown to inhibit lung metastasis in mouse osteosarcoma and melanoma. The CXCR1/CXCR2 antagonist-Resertaxin can inhibit the growth and metastasis of tumors in animal models and is used in clinical trials of patients with middle and advanced breast cancer. Therefore, the research on the effect of the chemotactic factor in the development of the tumor and the search of a new target point of the chemotactic factor for treating the breast cancer have important significance for explaining the tumor metastasis and wide drug market prospect.

The correlation of obesity with breast cancer was first seen in the 60's of the 20 th century, and case-control studies were the first to suggest women with a predisposition to postmenopausal obesity and hypertension for the onset of breast cancer. In animal experimental studies, it was also found that the tumor growth rate of obese mice was significantly increased compared to the control group. The world Foundation for cancer research and the American cancer research have conducted retrospective analysis of epidemiological evidence, and animal model studies have demonstrated that overweight and obesity can significantly increase the risk of breast cancer. Meta analysis shows that the risk of suffering from breast cancer is increased along with the increase of BMI, the risk of suffering from breast cancer of a patient with BMI more than 30kg/m2 is 1.3-2 times of that of a normal person, and the risk of suffering from breast cancer of a female can be obviously increased due to obesity.

The possible mechanism of obesity for promoting the occurrence and development of breast cancer comprises the following aspects of (1) estrogen. The obesity can increase the synthesis of estrogen, reduce the level of serum estradiol and Sex Hormone Binding Globulin (SHBG), and excess serum estrogen to compete for estrogen receptors on target organs, so as to cause hypothalamus-pituitary-ovarian gonadal axis dysfunction, break the balance of hyperplasia and involution of mammary gland tissues and promote the abnormal proliferation and transformation of breast cancer cells; (2) chronic inflammation. Activated macrophages in adipose tissues of obese patients can secrete a large amount of proinflammatory mediators to cause chronic inflammatory reaction, and a large amount of inflammatory factors and various cells in vivo form a tumor microenvironment for tumors, so that the generation and development of breast cancer cells are promoted. When the TG concentration in blood is too high, mammary vascular lipid deposition causes chronic inflammation, inflammatory factors secreted by inflammatory cells, interstitial fibroblasts, lymphocytes, macrophages, blood vessels, lymphatic vessel networks and extracellular matrix form a tumor microenvironment to interfere the replication and transcription of DNA (deoxyribonucleic acid), so that malignant tumors are generated; (3) insulin and insulin-like growth factor-1 (IGF-1). Obesity is prone to insulin resistance, increases insulin and IGF-1 levels, and contributes to cancer cell proliferation and angiogenesis. A study from the swiss database collected information on 115000 patients receiving insulin therapy in 2005 and found that patients treated with insulin glargine alone had a significantly higher risk of developing breast cancer over the next 2 years (RR ═ 1.99) than other insulin analogues. (4) An adipocyte cytokine. Obesity affects the production of adipocyte factors, reduces adiponectin, increases leptin and PAI-1 levels, promotes cell proliferation, resists apoptosis, and accelerates angiogenesis, thereby promoting the proliferation and growth of cancer cells. Adiponectin can inhibit mitosis promoted by cooperation of insulin and estrogen, and can also reduce metastasis and infiltration capacity of cancer cells by promoting expression of Bax and p53 apoptosis-promoting genes. Leptin can promote the growth of breast cancer by activating JAK/STAT3, MAPK-ERK1/2 or PI3K pathways; in addition, leptin can promote the generation of tumor-related blood vessels by inducing the expression of angiogenin, and leptin can also induce the transcription of human epidermal growth factor receptor 2ErbB-2), participate in the response of insulin-like growth receptor 1(IGF-1) in triple negative breast cancer cells, activate Epidermal Growth Factor Receptor (EGFR) and promote the invasion and metastasis of the cells. Obesity is a risk factor of postmenopausal breast cancer, the relationship between obesity and the occurrence and development of breast cancer is close and complex, and a large number of studies show that obesity can generate certain negative regulation and control on the occurrence, development, diagnosis, tumor characteristics and prognosis of breast cancer. Therefore, through further exploration and research on the relationship between the obesity, the adipose tissues and the adipose factors secreted by the adipose tissues and the breast cancer, a very important reference basis can be provided for the future formulation of corresponding measures for reducing the incidence rate of the breast cancer and improving the disease prognosis.

Adipose factor Chemerin

The adipokine Chemerin is also known as tazarotene inducible gene 2(TIG2) or retinol receptor responsive protein 2. After cloning by Nagpal et al in 1997, Wittamer et al isolated their active proteins by reverse phase high pressure liquid chromatography in ascites secondary to ovarian cancer in 2003. The Chemerin protein precursor has 163 amino acids in length, has 6 cysteine residues, forms 3 disulfide bonds, removes an N-terminal signal peptide and several C-terminal amino acids, has biological activity, has a structure in which an activated form of Chemerin in blood contains 134 amino acids (about 16kDa), belongs to the family of cecropin/cystatin, and can be rapidly converted into an activated form by several proteases when an inflammatory reaction occurs. Chemerin is widely expressed in various tissues of the human body, mainly in white adipose tissue, placenta and liver, and binds to 3 receptors: g protein coupled receptor CMKLR 1; ② G protein coupled receptor GPR 1; ③ CCRL-2. After Chemerin is combined with GPR1, on one hand, calcium ions in GPR1 positive cells can be released, cAMP aggregation is inhibited, and MAP kinase is phosphorylated; the other aspect has an effect of promoting the intracellular calcium ion influx. On one hand, Chemerin is used as a chemotactic factor to chemotact dendritic cells and macrophages, plays a bridge role between immunity and adaptive immunity, and chemotactic natural killer cells to reach inflammatory sites to participate in inflammatory reaction; on the other hand, as a novel adipokine, it is secreted and produced from adipose tissue, regulates differentiation and lipolysis of adipocytes, and promotes biological effects such as insulin signaling pathway in adipocytes. Chemerin plays an important role in the pathophysiological mechanisms of obesity and metabolic syndrome.

GPR1 receptor

GPR1 is the closest homolog to CMKLR1, sharing more than 40% sequence identity with CMKLR 1. It is a G protein-coupled receptor that plays a key role in eukaryotic cells, generally in the glucose stimulation, lipid accumulation and transmission of nutrient signals to the cAMP pathway. Many signal transduction is mediated by G proteins → coupled receptors (GPCRs) in eukaryotes. G protein β -coupled receptors are present in several eukaryotes, including yeast, dinoflagellates, and animals. GPR1 was originally a G protein-coupled receptor (GPCR) found in humans, which was identified as a receptor for chemerin by in vitro experiments. Although no physiological function of Chemerin binding to GPR1 was reported, GPR1 was reported to be highly expressed in brown adipose tissue, white adipose tissue and skeletal muscle in murine animals, and GPR1 was mainly expressed in vascular cells of white adipose tissue. Glucose intolerance was found to be more severe in GPR1 knockout mice fed a high fat diet than in WT mice. In addition, GPR1 knockout mice were able to inhibit glucose alpha stimulated elevation of insulin levels, resulting in elevated blood glucose, in pyruvate tolerance assays. These results indicate that GPR1 is an active receptor for Chemerin and can regulate glucose homeostasis during obesity.

Activity of CMKLR1 and GPR1 in HTLA cells following treatment with increasing Chemerin doses was measured using a Tango bioassay. Chemerin was found to activate CMKLR1 and GPR1 with similar efficacy, achieving maximal response (Emax) with 265.3 + -182.2-and 185.6 + -26.5-fold changes in effect. Furthermore, Chemerin has significantly higher potency (EC50,18.2 ± 2.9nM) for activating GPR1 than for activating CMKLR1(EC50,54.6 ± 30.7nM), demonstrating that Chemerin activates GPR1, while GPR1 is also a highly sensitive Chemerin receptor.

GPR1 expressing cell line by weak Ca²⁺Mobilization and activation of ERK1/2 respond to chemerin, but it is effectively internalized due to agonist binding. Thus, GPR1 may act as a decoy receptor for chemerin and, to date, GPR 1-mediated activity has not been described in major cells or in vivo. And GPR1 is expressed in the central nervous system, skeletal muscle, skin and adipose tissue, potentially modulating chemerin activity.

The G Protein Coupled Receptor Superfamily (GPCRs) play an important role in the process of transferring extracellular signals into cells, and regulate and control cell movement, growth and gene transcription, which are the vital factors in the biology of the three cancers. Over the past decade, mediated signaling pathways have been shown to be key regulators of proto-oncogene signaling and to be good drug targets, and 60% of the drugs currently on the market have been designed for this receptor. However, as one of the members of GPRs, GPR1 is not directly used for treating clinical cancer by any medicament at present in GPR 1. Therefore, the method has important social significance and wide economic market by taking GPR1 as a target and screening novel anti-cancer polypeptide drugs and humanized antibodies.

The Phage Display Technology (Phage Display Technology) is a screening Technology of specific polypeptide or protein, which can Display the polypeptide encoded by the target gene on the Phage surface in the form of fusion protein, the displayed polypeptide or protein can maintain relatively independent spatial structure and biological activity, and direct connection is established between a large number of random polypeptides and the DNA coding sequence thereof, so that the polypeptide ligands of various target molecules (such as antibody, enzyme, cell surface receptor and the like) can be rapidly identified by in vitro affinity panning procedure. Since the advent of phage display technology, phage antibody library technology has progressed very rapidly in recent decades due to its advantages of high library capacity, high efficiency, convenience, and flexible screening, and has gained wide application in many fields of life sciences, especially in the fields of tumor diagnosis and tumor antibody drug preparation, and has received increasing attention. At present, the main action target of the polypeptide drugs is G Protein Coupled Receptors (GPCRs), and 39 percent of polypeptide drugs target the GPCRs in clinical research. Polypeptide drugs have many advantages: the chemical composition has the advantages that the chemical composition is higher than that of common chemical medicaments, and the biological activity and the specificity are high; the toxic reaction is relatively weak, and accumulation is not easy to generate in vivo; and the interaction between the trioctyl phosphate and other medicines is less, and the affinity between the trioctyl phosphate and receptors in vivo is better. The humanized monoclonal antibody has the advantages of high sensitivity, strong specificity, high efficiency, little or no serum cross reaction, low preparation cost and the like. The integration of genotype and phenotype can be realized by screening polypeptide drugs and humanized antibodies by using phage display technology, and the technology is an efficient and practical technical method for preparing antibodies, screens out required protein or polypeptide by using the affinity of expressed protein and ligand, and is widely used for identifying and screening disease markers and screening and preparing antibody drugs.

Disclosure of Invention

In order to overcome the defects and shortcomings of the prior art, the primary object of the invention is to provide a GPR1 antagonistic polypeptide obtained by phage display library screening, the antagonistic polypeptide has specific high affinity with the Chemerin receptor GPR1, can block the signal path of Chemerin/GPR1 by inhibiting the binding of Chemerin and GPR1, and has great application value in the aspect of targeted therapy of diseases with high expression of GPR1 receptor.

Another object of the present invention is to provide derivatives of the above GPR1 receptor antagonist polypeptides, which derivatives also have specific high affinity for GPR1 receptor, specifically compete for binding sites of Chemerin to GPR1, and inhibit binding of Chemerin to GPR 1.

Still another object of the present invention is to provide the use of the GPR1 antagonist polypeptide and derivatives thereof as described above.

In order to realize the task, the invention adopts the following technical solution:

GPR1 receptor antagonistic polypeptide, characterized in that its amino acid sequence is Ala- (D) Asp-Ile-Arg-His-Ile-Lys-NH₂I.e., LRH 7-G2.

The method for screening the GPR1 antagonistic polypeptide utilizes a phage random peptide library, firstly transfects 293T cells with GPR1 plasmid to obtain a stable cell line permanently highly expressing GPR1, takes wild type 293T cells as control adsorption cells, carries out 5 rounds of whole cell subtractive screening, randomly picks 50 positive phage for amplification, extracts clone single-stranded DNA for sequencing. The basic characteristics of the amino acid sequence of the polypeptide are analyzed, polypeptide homology comparison is carried out, and a polypeptide motif with high occurrence frequency is searched. BLAST searches protein databases, detects proteins with high polypeptide sequence homology, finds biological species containing a large amount of the polypeptide, and possibly combined cell surface receptors and ligands, and is beneficial to subsequent extraction and purification of a large amount of polypeptide.

The hydrophilic analysis shows that LRH7-G2 is hydrophilic polypeptide;

the purity of the LRH7-G2 synthesis detected by High Performance Liquid Chromatography (HPLC) and Mass Spectrometry (MS) reaches 99.85%.

In another aspect, the derivative of the GPR1 receptor antagonist polypeptide of the present invention is a product obtained by conventionally modifying an amino acid side chain group of a GPR1 receptor antagonist polypeptide, an amino terminus or a carboxyl terminus of a GPR1 receptor antagonist polypeptide fragment, or a product obtained by attaching a tag for polypeptide or protein detection or purification to 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 and the like;

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

In a further aspect, the present invention provides a biologically active fragment or analogue of a GPR1 receptor antagonist polypeptide according to the present invention as set forth in SEQ ID nos. 2 to 7:

AX₁IRHIK SEQ ID No.2, wherein:

⑴X₁is glutamic acid (E).

2.ADX₂RHIK SEQ ID No.3, wherein:

⑴X₂is valine (V).

The invention also provides a derivative of the biological active fragment or analogue of the GPR1 receptor antagonist polypeptide, wherein the product is obtained by performing conventional modification on the amino acid side chain group, the amino terminal or the carboxyl terminal of the derivative of the biological active fragment or analogue of the GPR1 receptor antagonist polypeptide, or is obtained by connecting a label for polypeptide or protein detection or purification to the biological active fragment or analogue of the 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 and the like;

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

In the technical scheme of the invention, the GPR1 antagonist polypeptide and the derivative thereof can be applied to preparing medicaments for preventing and/or treating female reproductive diseases, and exist in the forms of: contains 6 amino acids; ② consists of 7 amino acids. The GPR1 antagonist polypeptide derivative is a product obtained by carrying out conventional modification on a side chain group of GPR1 antagonist polypeptide amino acid and an amino terminal or a carboxyl terminal of a GPR1 antagonist polypeptide fragment, or a product obtained by connecting a label for polypeptide or protein detection or purification to GPR1 antagonist polypeptide; the conventional modification is preferably amination, amidation, hydroxylation, carboxylation, carbonylation, alkylation, acetylation, phosphorylation, esterification, glycosylation, cyclization, biotinylation, fluorescent group modification, polyethylene glycol (PEG) modification or immobilization modification and the like; the tag is preferably His₆GST, EGFP, MBP, Nus, HA, IgG, FLAG, c-Myc, or ProfinityXact, etc.;

the GPR1 antagonist polypeptide or derivative thereof may be derived from a mammal or bird, such as a primate (human); rodents, including mice, rats, hamsters, rabbits, horses, cattle, canines, cats, and the like.

The GPR1 antagonist polypeptide derivative is preferably: the second amino acid residue of the GPR1 antagonistic polypeptide is D-configuration aspartic acid, and the tail end of the antagonistic polypeptide is amidated and modified to be Ala- (D) Asp-Ile-Arg-His-Ile-Lys-NH₂。

The GPR1 antagonistic polypeptide and the derivative thereof are obtained by adopting a known method in the prior art, and can be chemically synthesized by using an automatic polypeptide synthesizer; deducing a nucleotide sequence from the short peptide sequence, and cloning the nucleotide sequence into a vector for biosynthesis; it can also be extracted and purified in large quantities from existing organisms.

Specifically, GPR1 antagonist polypeptide LRH7-G2 and derivatives thereof have the following sequence:

1.AX₁IRHIK (SEQ ID No.2), wherein:

⑴X₁is glutamic acid (E).

2.ADX₂RHIK (SEQ ID No.3), wherein:

⑴X₂is valine (V).

In addition, GPR1 antagonist polypeptide LRH7-G2 and derivatives thereof also include the following naturally occurring polypeptides in organisms:

in a further aspect, the invention provides a GPR1 receptor antagonist polypeptide, a GPR1 receptor antagonist polypeptide derivative, a GPR1 receptor antagonist polypeptide bioactive fragment or analog and a derivative thereof, which are provided by the invention, and application of the GPR1 receptor antagonist polypeptide, the GPR1 receptor antagonist polypeptide derivative, the GPR1 receptor antagonist polypeptide bioactive fragment or analog and the derivative thereof in preparation of medicines for treating chemerin-GPR1 mediated diseases.

In a further aspect, the invention provides a GPR1 receptor antagonist polypeptide, a derivative of a GPR1 receptor antagonist polypeptide, a biologically active fragment or analog of a GPR1 receptor antagonist polypeptide, and derivatives thereof as described herein for use in the treatment of a chemerin-GPR1 mediated disease.

In the technical scheme of the invention, the chemerin-GPR1 mediated disease is selected from breast cancer, fatty liver, diabetes, inflammatory reaction and polycystic ovary syndrome.

In a further aspect, the invention provides a GPR1 receptor antagonist polypeptide, a GPR1 receptor antagonist polypeptide derivative, a GPR1 receptor antagonist polypeptide bioactive fragment or analog and a derivative thereof, which are provided by the invention, and application of the GPR1 receptor antagonist polypeptide, the GPR1 receptor antagonist polypeptide derivative, the GPR1 receptor antagonist polypeptide bioactive fragment or analog and the derivative thereof in preparation of a medicine for inhibiting reduction of cAMP concentration caused by chemerin.

In a further aspect, the invention provides the use of a GPR1 receptor antagonist polypeptide, a derivative of a GPR1 receptor antagonist polypeptide, a biologically active fragment or analog of a GPR1 receptor antagonist polypeptide, and derivatives thereof of the present invention for inhibiting a decrease in cAMP concentration caused by chemerin.

In still another aspect, the invention provides a GPR1 receptor antagonist polypeptide, a GPR1 receptor antagonist polypeptide derivative, a GPR1 receptor antagonist polypeptide bioactive fragment or analog, and a GPR1 receptor antagonist polypeptide derivativePreparation of calcium (Ca) for inhibiting chemerin-induced calcium²⁺) The use in medicine for internal flow action.

In still another aspect, the invention provides a GPR1 receptor antagonist polypeptide, a GPR1 receptor antagonist polypeptide derivative, a GPR1 receptor antagonist polypeptide bioactive fragment or analog and a GPR1 receptor antagonist polypeptide derivative for inhibiting chemerin-induced calcium (Ca)²⁺) Use in an influx effect.

In a further aspect, the invention provides a GPR1 receptor antagonist polypeptide, a GPR1 receptor antagonist polypeptide derivative, a GPR1 receptor antagonist polypeptide bioactive fragment or analogue and a derivative thereof, which are provided by the invention, in application to preparation of a medicament for inhibiting cell chemotaxis caused by chemerin.

In a further aspect, the invention provides a GPR1 receptor antagonist polypeptide, a GPR1 receptor antagonist polypeptide derivative, a GPR1 receptor antagonist polypeptide bioactive fragment or analog and a derivative thereof, which are provided by the invention, and application of the GPR1 receptor antagonist polypeptide, the GPR1 receptor antagonist polypeptide derivative, the GPR1 receptor antagonist polypeptide bioactive fragment or analog and the derivative thereof in inhibition of cell chemotaxis caused by chemerin.

In still another aspect, the invention provides the use of the GPR1 receptor antagonist polypeptide, the GPR1 receptor antagonist polypeptide derivative, the GPR1 receptor antagonist polypeptide biologically active fragment or analog, and the GPR1 receptor antagonist polypeptide derivative of the present invention in the preparation of a medicament for the treatment of breast cancer, fatty liver, diabetes, inflammatory response, polycystic ovary syndrome.

In a further aspect, the invention provides the use of a GPR1 receptor antagonist polypeptide, a GPR1 receptor antagonist polypeptide derivative, a GPR1 receptor antagonist polypeptide biologically active fragment or analog and derivatives thereof of the present invention for the treatment of breast cancer, fatty liver, diabetes, inflammatory responses, polycystic ovary syndrome.

In a further aspect, the invention provides a pharmaceutical composition comprising as an active ingredient one or more of a GPR1 receptor antagonist polypeptide, a GPR1 receptor antagonist polypeptide derivative, a GPR1 receptor antagonist polypeptide biologically active fragment or analog, and derivatives thereof as described herein.

The pharmaceutical composition may contain one or more 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, a lubricant, or the like;

the pharmaceutical composition can be further prepared into various forms such as tablets, granules, capsules, oral liquid or injection, and the like, and the medicines of various formulations can be prepared according to the conventional method in the pharmaceutical field;

the invention also discloses a medicament for preventing and/or treating female reproductive diseases or tumors, which comprises at least one of the GPR1 receptor antagonist polypeptide, a GPR1 receptor antagonist polypeptide derivative, a GPR1 receptor antagonist polypeptide bioactive fragment or analogue and a derivative thereof;

the invention also discloses a medicament for preventing and/or treating diseases with high GPR1 receptor expression, which comprises at least one of GPR1 receptor antagonist polypeptide, GPR1 receptor antagonist polypeptide derivatives, GPR1 receptor antagonist polypeptide bioactive fragments or analogues and derivatives thereof.

In a specific experiment, the obtained GPR1 antagonistic polypeptide LRH7-G2 can effectively relieve the inhibitory effect of chemerin on a cAMP signal path. Wherein, the GPR1 antagonistic polypeptide LRH7-G2 derivatives (SEQ ID No. 1-7) have the same effect.

In another specific experiment of the invention, GPR1 antagonistic polypeptide LRH7-G2 can be used for effectively inhibiting chemerin-induced calcium (Ca)²⁺) And (4) internal flow effect. Wherein, the GPR1 antagonistic polypeptide LRH7-G2 derivatives (SEQ ID No. 1-7) have the same effect.

Compared with the prior art, the invention has the following advantages and effects:

(1) the invention provides a GPR1 antagonistic polypeptide LRH7-G2 and a derivative (SEQ ID No. 1-7) thereof, wherein the antagonistic polypeptide and the derivative thereof can be specifically combined with GPR1, specifically compete a binding site of Chemerin and GPR1, and can inhibit a Chemerin/GPR1 signal channel. The biological polypeptide medicament serving as a binding site of Chemerin/GPR1 can be used for preparing medicaments for preventing and/or treating diseases with high expression of GPR1 receptor, such as: breast cancer, fatty liver, diabetes, and inflammatory reaction, polycystic ovary syndrome. Can be widely applied in the medical and biological fields and can generate huge social and economic benefits.

Drawings

FIG. 1: high Performance Liquid Chromatography (HPLC) assay format for LRH 7-G2.

FIG. 2: LRH7-G2 Mass Spectrometry (MS) detection.

FIG. 3: hydrophobic profile comparison analysis of GPR1, chemerin and LRH7-G2 polypeptides. Wherein: a: GPR1 hydrophobic profile; b: chemerin hydrophobic profile; c: LRH7-G2 polypeptide hydrophobic profile; d: hydrophobic profile comparison plots of GPR1, chemerin and LRH7-G2 polypeptides. GPR1 profile is red, chemerin profile is blue, LRH7-G2 profile is green;

FIG. 4: LRH7-G2 alleviated the inhibitory effect of chemerin on the cAMP signaling pathway. Among them, the role of A, LRH7-G2 in 293T wild-type cells together with Forsklin and Chemerin; b, LRH7-G2 alone in 293T GPR1^+/+The function of (1); c, LRH7-G2 with Forsklin and Chemerin in 293T GPR1^+/+The function of (1);

FIG. 5: LRH7-G2 inhibited chemerin-induced calcium (Ca)²⁺) And (4) internal flow effect.

Detailed Description

In order that the invention may be more clearly understood, it will now be further described with reference to the following examples and the accompanying drawings. The examples are for illustration only and do not limit the invention in any way. In the examples, each raw reagent material is commercially available, and the experimental method not specifying the specific conditions is a conventional method and a conventional condition well known in the art, or a condition recommended by an instrument manufacturer.

Example 1: and carrying out panning, amplification, purification, sequencing and synthesis on GPR1 antagonistic polypeptide LRH 7-G2.

The embodiment mainly aims to obtain a positive phage specifically bound with GPR1 through screening, amplifying and purifying the positive phage, extracting phage single-stranded DNA (ssDNA) for sequencing, analyzing and comparing the obtained sequences, and finally synthesizing the high-purity antagonist polypeptide LRH 7-G2.

The method comprises the following specific steps:

1. establishment of a 293T cell line permanently overexpressing GPR 1: 293T-GPR1^+/+/LRH

Selecting luminous human 293T cell with vigorous growth, and culturing at 5X 10/day before transfection⁵One/well, inoculating in 6-well plate, culturing until the cell fusion degree is 60% after the second day;

② the second day, with 6 hole plate culture hole as a unit, using 200 u L of opti-MEM medium dilution 3 u g plasmid, another 200 u L opti-MEM medium dilution 6 u L liposome Lipofectamine2000, after gently mixing, placed at room temperature for 5 minutes;

③ mixing the two tube dilutions gently, standing for 20 minutes at room temperature, and then adding 600 μ L of opti-MEM culture medium gently into the mixed dilutions;

rinsing the cells to be transfected with PBS slightly once, then adding the mixed diluent into the culture holes slightly, and culturing in a carbon dioxide incubator;

fifthly, after culturing for 4-6 hours, abandoning the culture medium used for transfection, and adding 3mL of complete culture medium into the hole;

sixthly, selecting a culture medium containing 1 mu g/mL puromycin (puromycin) for screening after 48 hours; A293T cell line stably expressing GPR1 was obtained after the cells no longer died.

Seventhly, extracting total RNA by using TRIzol, quantifying 2 mug of RNA for reverse transcription (a reverse transcription kit, purchased from Promega corporation), and performing qPCR by using a specific primer sequence.

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

Fw 5’-AATGCCATCGTCATTTGGTT-3’(SEQ ID No.8)

Rv 5’-CAACTGGGCAGTGAAGGAAT-3’(SEQ ID No.9)

(viii) comparing with transfected pSM2c-Hu-scramble RNA, detecting high expression level of GPR1 and naming as: 293T-GPR1^+/+LRH, i.e., can be used for positive phage selection.

2. The GPR1 antagonist polypeptide was subjected to panning, amplification, purification, sequencing and synthesis.

Preparation of ER2738 host bacterial liquid: sterileThe technical operation is that 200 mul LB-Tet liquid culture medium is taken firstly in a 1.5ml sterilized centrifuge tube, then 0.2 mul bacterial liquid is taken from the glycerol frozen stock of E.coli ER2738 and is fully and evenly mixed with the liquid, all the liquid is absorbed and coated on an LB-Tet flat plate, the flat plate is marked, the flat plate is placed for 3min at room temperature, and then the flat plate is placed in a 37 ℃ constant temperature incubator for inversion overnight culture. Observing the next day, sealing with sealing film after clone grows out, and storing at 4 deg.C in dark for use. Single colonies were picked by aseptic technique using a sterilized tip, placed in 10ml sterilized centrifuge tubes previously supplemented with 3ml LB-Tet broth, labeled and cultured overnight on a constant temperature shaker at 37 ℃ under shaking at 300 rpm/min. The next day, the bacterial amplification solution was stored at 4 ℃ for future use. Taking 10ml of a sterilized centrifuge tube, adding 3ml of LB-Tet liquid culture medium in a sterile operation, inoculating 30 mu l of overnight-cultured bacteria, carrying out shake culture at constant temperature of 37 ℃ and 300rpm/min for 2-3 h, wherein the bacteria are in an exponential growth phase and are in a fog shape (OD) by visual observation₆₀₀～0.5)。

GPR1 panning of antagonistic peptides: high expressing GPR1 cells as 10⁵The culture dish is inoculated on 60X 15mm which is coated with polylysine in advance²In a culture dish, when the cells are cultured to 80-90% of the cell density, 1 mu l of eluent is firstly taken for each round of elutriation (meanwhile, a cell line which does not express GPR1 is used as a blank control), the rest eluent is added into 20ml of LB culture solution for amplification, then purification and the titer after amplification are detected again, the amplification product is stored for a short time at 4 ℃, the same number of orders are taken for the next round of elutriation, and the rest amplification product is stored at-20 ℃ by using 50% of glycerol.

And thirdly, measuring the titer of the phage, namely taking 4 sterilized 10ml centrifuge tubes, preparing 1 sterilized centrifuge tube for each phage dilution, melting Top agar (agar Top) by a microwave oven, adding 3ml Top agar into each tube, and carrying out water bath at 45 ℃ for later use. For each dilution of phage, 1 LB/IPTG/Xgal plate was prepared and pre-warmed in a 37 ℃ incubator for use. Will OD₆₀₀Coli ER2738 E.coli 0.5 was aliquoted at 200. mu.l phage dilution per tube and stored at 4 ℃ until use. Taking 4 sterilized 1.5ml centrifuge tubes, respectively containing 100. mu.l, 90. mu.l LB-Tet culture medium, sucking 1. mu.l of bacteriophage to be tested into 100. mu.l LB-Tet culture medium, diluting according to 10-fold gradient, respectively marking as 10^-1、10^-2、10^-3、10^-4And each dilution is mixed evenly by gentle oscillation and then is centrifuged instantly. Mix 10 μ l of each dilution of phage to be titrated with 200 μ l of E.coli ER2738, mix by gentle shaking, centrifuge instantaneously, incubate for 5min at room temperature. Quickly adding the mixed bacterial liquid into top agar, quickly shaking and uniformly mixing, immediately pouring into a preheated LB/IPTG/Xgal plate, uniformly flattening, cooling at room temperature for 5min, and inversely culturing the plate in a constant-temperature incubator at 37 ℃ overnight.

Amplification and purification of eluted phage: taking a 250ml conical flask, adding the overnight cultured ER2738 host bacterial liquid into 20ml of LB liquid culture medium according to the proportion of 1:100, and carrying out vigorous shaking culture at 37 ℃ and 250rpm for 2 h; then adding the phage liquid to be amplified into an erlenmeyer flask, and carrying out vigorous shaking culture at 37 ℃ and 250rpm for 4.5 h; the culture was transferred to a 50ml centrifuge tube and centrifuged at 10,000rpm at 4 ℃ for 10 min. Transferring the supernatant into another clean centrifugal tube, and centrifuging again at 10,000rpm at 4 ℃ for 10 min; transferring 80% of the supernatant into another clean centrifuge tube, adding 1/4 volume of PEG/NaCl, reversing, mixing uniformly, and precipitating at 4 ℃ overnight; the next day, the pellet was centrifuged at 12,000rpm for 20min at 4 ℃. Carefully sucking the supernatant with a clean gun head, centrifuging at 4 deg.C and 12,000rpm for 1min, and removing the residual supernatant; the pellet was then resuspended in 1ml TBS and gently pipetted 100 times. Then transferring the suspension into a 2ml centrifuge tube, and centrifuging at 4 ℃ and 10,000rpm for 5min to remove residual cells; adding 1/4 volume of PEG/NaCl to the supernatant, and incubating on ice for 60min for reprecipitation; taking out the centrifuge tube, centrifuging at 4 deg.C and 12,000rpm for 20min, and removing supernatant; the pellet was resuspended in 200. mu.l TBS and centrifuged at 10,000rpm for 1min at 4 ℃. The supernatant was transferred to another centrifuge tube. Short-term storage at 4 deg.C, or long-term storage at-20 deg.C with 50% glycerol. The amplification of the monoclonal phage comprises the steps of adding overnight cultured ER2738 host bacterial liquid into 2mL of LB liquid culture medium according to the proportion of 1:100, and carrying out vigorous shaking culture at 37 ℃ and 250rpm for 2 h; selecting a plate with less than 100 plaques from the fourth round of titer plates by using a sterilizing toothpick, picking well-separated blue plaques, adding the blue plaques into a culture tube, and carrying out violent shake culture at 37 ℃ and 250r/min for 4.5 h; the culture was then transferred to a fresh centrifuge tube and centrifuged at 10,000rpm for 30sec at 4 ℃. Transferring the supernatant into a fresh tube, and centrifuging once again; 80% of the supernatant was transferred to fresh centrifuge tubes and stored at 4 ℃ or stored with 50% glycerol for a long period at-20 ℃.

Identifying M13 bacteriophage ssDNA by agarose gel electrophoresis: horizontally placing a gel forming mold, placing the selected comb, and reserving a space of 1mm between the bottom of the comb and the mold; weighing 1g of agarose for DNA electrophoresis, putting the agarose into a 250ml triangular flask, adding 100ml of 1 XTAE buffer solution, uniformly mixing, putting the flask into a microwave oven, heating and boiling until the agarose is completely dissolved; and (3) closing the induction cooker, taking out the triangular flask, cooling the triangular flask to room temperature (which can be tolerated by holding the flask by hand), adding 5 mu l of ethidium bromide, and pouring the gel solution into a rubber plate paving plate after uniform mixing. The rubber plate used in the experiment needs about 100ml of rubber solution; after the gel is completely solidified at room temperature and takes about 30 minutes, pulling out the comb teeth, and putting the rubber plate into an electrophoresis tank; adding 1 XTAE buffer solution into the electrophoresis tank, preferably 2mm higher than the surface of the gel; diluting the sample with a Loading buffer, adding the diluted sample into a gel plate, and paying attention to that a suction head of a sample injector is just placed in a gel point sample hole, the gel cannot be punctured, and the sample is prevented from overflowing out of the hole; switching on a power supply, adjusting the voltage to 50V, performing electrophoresis for 90min, taking out the gel plate, and observing the result under an ultraviolet lamp.

Sequencing and sequence analysis of ssDNA: the extracted M13 phage ssDNA was sent to Shanghai Yingji Biotechnology Ltd for DNA sequencing. Sequencing was followed by sequence analysis using Bioedit software. According to the analysis result, the sequence of the sample is Ala- (D) Asp-Ile-Arg-His-Ile-Lys-NH₂Wherein the second aspartic acid is in D configuration, represented by LRH7-G2, and finally the short peptide is synthesized by Shanghai Qiaozao organism.

The purity of the LRH7-G2 polypeptide detected by High Performance Liquid Chromatography (HPLC) in FIG. 1 and Mass Spectrometry (MS) in FIG. 2 was 99.85%, and the molecular weight was consistent with the predicted value. The hydrophobic profile analysis of FIG. 3 shows that the LRH7-G2 polypeptide has a certain similarity to chemerin, and the similarity reaches 0.0061350(PAM 250).

Example 2GPR1 antagonistic polypeptide LRH7-G2 can effectively relieve the inhibitory effect of chemerin on cAMP signaling pathway.

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

plating cells: wild type 293T cells and 293T cells with high expression of GPR1 (293T GPR 1)^+/+) At 5X 10, respectively⁵One cell/well was inoculated in 6-well cell culture plates with a medium volume of 1mL per well, placed in an incubator for 24h, starved overnight, and treated with different concentration gradients (3. mu.M, 0.3. mu.M, 0.03. mu.M) of LRH7-G2 polypeptide, Fosklin (25. mu.M) and chemerin (30nM) for 6 h;

preparing a sample: adding 300 mu L of cell lysate into each hole, standing at 4 ℃ for 20 minutes, scraping and collecting cells by using a cell scraper, turning upside down and uniformly mixing, centrifuging at 12,000rpm for 10 minutes, and collecting supernatant;

③ measuring the concentration of the sample: the sample concentration was determined by BCA method;

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

a, preparing required reagents, and preparing 3 multiple wells for each sample;

b, adding 50 mu L of sample or standard substance into a 96-well plate coated with the antibody; then 25 μ L of cAMP peroxidase tracer conjugate was added to each well;

c, adding 50 mu L of Anti-cAMP antibody into each hole, slowly incubating for 30 minutes on a shaking table at room temperature;

d, washing 5 times by using eluent, adding 100 mu L of chemical light-reflecting agent into each hole, and incubating for 5 minutes at room temperature;

and e, reading the plate by the microplate reader, and recording the luminescence value.

FIG. 4 shows that chemerin can reduce cellular cAMP concentration at a concentration of 30nM, but in 293T cells highly expressing GPR1 (293T GPR 1)^+/+) When LRH7-G2 (3. mu.M, 0.3. mu.M, 0.03. mu.M) was added at different concentrations, the cAMP concentration was significantly increased. In wild 293T cells, the GPR1 receptor is not expressed, so LRH7-G2 has no obvious inhibition effect on the effect of chemerin on reducing cAMP concentration. Similarly, in the single-action group of LRH7-G2 short peptide, LRH7-G2 did not increase cAMP concentration. From the above, it was concluded that LRH7-G2 specifically inhibited further the decrease of cAMP concentration by chemerin by acting with GPR 1. P<0.05,**P<0.01,***P<0.001 compared to the Forskolin + chemerin group.

Example 3GPR1 antagonist polypeptide LRH7-G2 can effectively inhibit chemerin-induced calcium (Ca)²⁺) And (4) internal flow effect.

Plating cells: wild type 293T cells and 293T cells with high expression of GPR1 (293T GPR 1)^+/+) At 5X 10, respectively³Inoculating each cell/well into a 96-well cell culture plate, wherein the volume of a culture medium in each well is 200 mu L, placing the cell culture plate in an incubator for 24 hours, and then starving the cell culture plate overnight;

preparing a reagent: dissolving probenecid into 1mL of buffer solution to prepare probenecid with the concentration of 250nM, shaking up, and adding into a fluorescent reagent for later use;

③ removing the cell culture medium, adding LRH7-G2 polypeptide with different concentration gradients (30. mu.M, 3. mu.M, 0.3. mu.M, 0.03. mu.M, 0.003. mu.M) and chemerin (0.3nM) for 30 minutes, and then adding 100. mu.L of the above fluorescent reagent into each well;

fourthly, placing the mixture for 30 minutes at 37 ℃ and then placing the mixture for 30 minutes at room temperature;

measuring fluorescence absorbance at 494nm for exciting light and 516nm for emitting light.

FIG. 5 shows the expression of GPR1 in 293T cells (293T GPR 1)^+/+) In the case of chemerin, calcium (Ca) was promoted at an action concentration of 0.3nM²⁺) Flowing signal pathway, increasing calcium ion (Ca)²⁺) And (4) concentration. After different concentrations of LRH7-G2 short peptide are added, calcium ions (Ca) can be remarkably reduced²⁺) Concentration, inhibition of chemerin on calcium (Ca)²⁺) Activation of the flow signal path. However, chemerin is on calcium (Ca) in wild type 293T cells not expressing GPR1²⁺) The signaling pathway of the flow is inactive, and LRH7-G2 is calcium (Ca) antagonist²⁺) The flow signaling pathway also had no effect, and it was concluded from this experiment that chemerin can activate calcium (Ca) by binding to the receptor GPR1²⁺) The signal path is flowed, and the LRH7-G2 short peptide can specifically inhibit the chemerin/GPR1 signal path to reduce calcium ions (Ca)²⁺) And (4) concentration. P<0.05,**P<0.01,***P<0.001 compared to the chemerin group.

SEQUENCE LISTING

<110> Shenzhen advanced technology research institute

<120> GPR1 antagonistic polypeptide, and derivative and application thereof

<130> CP11701248C

<160> 9

<170> PatentIn version 3.3

<210> 1

<211> 7

<212> PRT

<213> Artificial sequence

<400> 1

Ala Asp Ile Arg His Ile Lys

1 5

<210> 2

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (2)..(2)

<223> glutamic acid

<400> 2

Ala Xaa Ile Arg His Ile Lys

1 5

<210> 3

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (3)..(3)

<223> valine

<400> 3

Ala Asp Xaa Arg His Ile Lys

1 5

<210> 4

<211> 7

<212> PRT

<213> fungus, bacterium or alga

<400> 4

Ala Asp Ile Arg His Ile Lys

1 5

<210> 5

<211> 7

<212> PRT

<213> fungus, bacterium or alga

<400> 5

Ala Asp Ile Arg His Ile Lys

1 5

<210> 6

<211> 6

<212> PRT

<213> bacteria, fungi, fish, cabbage mustard, butterfly, peanut

<400> 6

Asp Ile Arg His Ile Lys

1 5

<210> 7

<211> 6

<212> PRT

<213> bacteria, fungi, fish, cabbage mustard, butterfly or peanut

<400> 7

Asp Ile Arg His Ile Lys

1 5

<210> 8

<211> 20

<212> DNA

<213> Artificial sequence

<400> 8

aatgccatcg tcatttggtt 20

<210> 9

<211> 20

<212> DNA

<213> Artificial sequence

<400> 9

caactgggca gtgaaggaat 20

Claims (13)

1. A GPR1 antagonist polypeptide, which has an amino acid residue sequence of Ala- (D) Asp-Ile-Arg-His-Ile-Lys-NH₂。

2. A derivative of a GPR1 antagonist polypeptide according to claim 1, characterized in that:

the derivative is a product obtained by connecting GPR1 antagonistic polypeptide with a label for polypeptide or protein detection or purification;

the label is His₆GST, EGFP, MBP, Nus, HA, IgG, FLAG, c-Myc or ProfinityXact.

3. A polynucleotide encoding a GPR1 antagonist polypeptide of claim 1.

4. A vector comprising the polynucleotide of claim 3 linked to a promoter sequence by gene technology means.

5. A host cell transfected with the vector of claim 4.

6. Use of a GPR1 receptor antagonist polypeptide according to claim 1 or a derivative according to claim 2 for the manufacture of a medicament for the treatment of a chemerin-GPR 1-mediated disease.

7. The use of claim 6, wherein the chemerin-GPR 1-mediated disease is selected from the group consisting of breast cancer, fatty liver, diabetes, inflammatory response, polycystic ovary syndrome.

8. Use of a GPR1 receptor antagonist polypeptide according to claim 1 or a derivative according to claim 2 for the preparation of a medicament for inhibiting a decrease in cAMP concentration caused by chemerin.

9. Use of a GPR1 receptor antagonist polypeptide according to claim 1 or a derivative according to claim 2 for the production of a medicament for inhibiting chemerin-induced calcium influx.

10. Use of a GPR1 receptor antagonist polypeptide according to claim 1 or a derivative according to claim 2 for the preparation of a medicament for inhibiting chemotaxis of cells caused by chemerin.

11. A pharmaceutical composition comprising as an active ingredient one or more of a GPR1 receptor antagonist polypeptide of claim 1, a derivative of claim 2.

12. The pharmaceutical composition of claim 11, wherein the pharmaceutical composition comprises one or more pharmaceutically acceptable carriers.

13. The pharmaceutical composition of claim 12, wherein the pharmaceutically acceptable carrier is a diluent, excipient, filler, binder, wetting agent, disintegrant, absorption enhancer, adsorptive carrier, surfactant, or lubricant; the pharmaceutical composition is further prepared into dosage forms of tablets, granules, capsules, oral liquid or injections, and the pharmaceutical compositions of various dosage forms are prepared according to conventional methods in the pharmaceutical field.

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