WO2019119273A1 - 一种gpr1拮抗多肽及其衍生物与应用 - Google Patents

一种gpr1拮抗多肽及其衍生物与应用 Download PDF

Info

Publication number
WO2019119273A1
WO2019119273A1 PCT/CN2017/117256 CN2017117256W WO2019119273A1 WO 2019119273 A1 WO2019119273 A1 WO 2019119273A1 CN 2017117256 W CN2017117256 W CN 2017117256W WO 2019119273 A1 WO2019119273 A1 WO 2019119273A1
Authority
WO
WIPO (PCT)
Prior art keywords
gpr1
antagonist polypeptide
derivative
polypeptide
chemerin
Prior art date
Application number
PCT/CN2017/117256
Other languages
English (en)
French (fr)
Inventor
张键
代小勇
汪宝蓓
张居作
赵华山
王鹤霏
Original Assignee
深圳先进技术研究院
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 深圳先进技术研究院 filed Critical 深圳先进技术研究院
Priority to PCT/CN2017/117256 priority Critical patent/WO2019119273A1/zh
Publication of WO2019119273A1 publication Critical patent/WO2019119273A1/zh

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/08—Peptides having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids

Definitions

  • the present invention relates to the fields of biotechnology and biomedicine, and in particular, the present invention is a target of female reproductive disease GPR1 receptor antagonist polypeptide LRH7-G1 and its derivatives and applications.
  • 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.
  • Metastasis to lymphatics after breast cancer surgery is a common metastatic symptom of breast cancer.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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%.
  • EGFR By activating downstream Ras/Raf/MEK/ERK and PI3K/AKT signaling pathways, EGFR promotes breast cancer proliferation, migration and Invasion [39,40];
  • epidermal growth factor receptor 2 HER-2
  • HER-2 epidermal growth factor receptor 2
  • PI3K/Akt PI3K/Akt
  • Ras/MAPK mitogen activator-associated kinase
  • Signaling pathways promote breast cancer proliferation, angiogenesis, migration and invasion.
  • 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.
  • trastuzumab for epidermal growth factor receptor-2 treatment against everolimus, an inhibitor in the PI3K/AKT/mTOR pathway
  • anti-angiogenesis Drug bevacizumab against BRCA1/2 mutation
  • palbociclib against CDK4/6 inhibitor palbociclib against CDK4/6 inhibitor.
  • 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.
  • 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.
  • CAFs Tumor-associated fibroblasts
  • 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.
  • treatment with an oncolytic virus carrying a CXCR4 antagonist inhibits angiogenesis and tumor metastasis.
  • 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 [63].
  • 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.
  • Estrogen 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;
  • chronic inflammation Activated macrophages in adipose tissue of obese patients can secrete a large number of pro-inflammatory mediators, causing chronic inflammatory reactions.
  • 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).
  • 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.
  • IGF-1 insulin-like growth receptor 1
  • 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.
  • Chemerin is also known as tazarotene-inducible gene 2 (TIG2) or retinol receptor-reactive protein 2.
  • TAG2 tazarotene-inducible gene 2
  • retinol receptor-reactive protein 2 retinol receptor-reactive protein 2.
  • the form contains 134 amino acids (about 16 kDa) and is structurally a family of bactericidal peptides/cysteine protease inhibitors.
  • 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.
  • 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 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.
  • GPCR G protein-coupled receptor
  • GPR1 is an active receptor for Chemerin that regulates glucose homeostasis during obesity.
  • CMKLR1 and GPR1 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.
  • GPCRs G-protein coupled receptor superfamilies
  • 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.
  • GPCRs G-protein coupled receptors
  • 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.
  • 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 blocks the signaling pathway of Chemerin/GPR1, which has great application value in the targeted therapy of diseases with high expression of GPR1 receptor.
  • 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.
  • the screening method of the above GPR1 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, and performs 5 rounds of whole cells.
  • the screening was reduced, 50 positive phage amplifications were randomly picked, and cloned single-stranded DNA sequencing was performed.
  • 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.
  • 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.
  • 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;
  • the tag is His6, GST, EGFP, MBP, Nus, HA, IgG, FLAG, c-Myc or ProfinityeXact.
  • 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;
  • 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;
  • the tag is His6, GST, EGFP, MBP, Nus, HA, IgG, FLAG, c-Myc or ProfinityeXact.
  • the GPR1 antagonist polypeptide and the derivative thereof can be applied to the preparation of a medicament for preventing and/or treating female reproductive diseases, and the present invention has the following forms: 1 contains 6 amino acids; 2 consists of 7 amino acids composition.
  • 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.
  • 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 mammals or birds, such as primates (humans); rodents, including mice, rats, hamsters, rabbits, horses, cattle, dogs. , cats, etc.
  • the derivative of the GPR1 antagonist polypeptide is: the second amino acid residue of the GPR1 antagonist polypeptide is a D-configuration tryptophan, and the terminal is amidated, that is, Ala-(D)Trp-Pro-Tyr-Val -Thr-Leu-NH 2 .
  • 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.
  • GPR1 antagonist polypeptide LRH7-G1 and its derivatives include the following polypeptides naturally present in the organism:
  • a further aspect of the invention provides a polynucleotide encoding the polypeptide of any one of SEQ ID No. 1-5.
  • a vector comprising a nucleotide of the invention which can be linked to a promoter sequence by genetic means.
  • a host cell transfected with a vector of the 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 chemotactic hormone. Use in GPR1 mediated diseases.
  • a GPR1 receptor antagonist polypeptide for treating a chemokine GPR1 Use in guiding diseases.
  • the chemokine-GPR1 mediated disease is selected from the group consisting of breast cancer, fatty liver, diabetes, inflammatory response, and polycystic ovary syndrome.
  • 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 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 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.
  • 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.
  • the present invention utilizes the obtained GPR1 antagonist polypeptide LRH7-G1 to effectively alleviate the inhibitory effect of chemerin on cAMP signaling pathway.
  • the GPR1 antagonist polypeptide LRH7-G1 derivative (SEQ ID No. 1 to 5) has the same effect.
  • the GPR1 antagonist polypeptide LRH7-G1 can effectively inhibit the influx of calcium (Ca 2+ ) induced by chemerin.
  • the GPR1 antagonist polypeptide LRH7-G1 derivative (SEQ ID No. 1 to 5) has the same effect.
  • the present invention provides a GPR1 antagonist polypeptide LRH7-G1 and a derivative thereof (SEQ ID No. 1 to 5), which are capable of specifically binding to GPR1 and specifically competing Chemerin binds to GPR1 and inhibits the Chemerin/GPR1 signaling pathway. It can be used as a bio-based peptide drug for Chemerin/GPR1 binding site, and can be used for preparing drugs for preventing and/or treating diseases high in expressing GPR1 receptor, for example, breast cancer, fatty liver, diabetes, inflammatory reaction, polycystic ovary synthesis disease. It can be widely used in the fields of medicine and biology, and it has enormous social and economic benefits.
  • Figure 1 LRH7-G1 high performance liquid chromatography (HPLC) detection chart.
  • Figure 2 LRH7-G1 mass spectrometry (MS) detection map.
  • Figure 3 Comparative analysis of hydrophobic profiles of GPR1, chemerin and LRH7-G1 polypeptides. Among them: A: GPR1 hydrophobic profile; B: chemerin hydrophobic profile; C: LRH7-G1 polypeptide hydrophobic profile; D: GPR1, chemerin and LRH7-G1 polypeptide hydrophobic profile comparison chart.
  • the GPR1 outline is red, the chemerin outline is blue, and the LRH7-G1 outline is green;
  • LRH7-G1 attenuates the inhibitory effect of chemerin on the cAMP signaling pathway.
  • A LRH7-G1 and Forsklin and Chemerin in 293T wild-type cells
  • B LRH7-G1 alone in 293T GPR1 + / +
  • C LRH7-G1 with Forsklin and Chemerin at 293T GPR1 + / The role of +
  • Figure 5 LRH7-G1 inhibits the influx of calcium (Ca 2+ ) induced by chemerin.
  • Example 1 Panning, amplification, purification, sequencing and synthesis of GPR1 antagonist polypeptide LRH7-G1.
  • 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.
  • 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;
  • 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;
  • a medium containing 1 ⁇ g/mL puromycin was selected for screening; a 293T cell line stably expressing GPR1 was obtained after the cells no longer died.
  • the specific primer sequence used is the Hu-GPR1 primer sequence:
  • 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 1 ⁇ l of titer for each round of eluate, and add the remaining to 20ml of LB medium for amplification, purification, and finally measurement.
  • 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.
  • 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 ⁇ l/tube, and stored at 4 ° C until use.
  • 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.
  • the supernatant was transferred to another clean centrifuge tube and 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 transferred to a 2 ml centrifuge tube, and the residual cells were removed by centrifugation at 10,000 rpm for 5 min at 4 ° C; 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 taken out at 4 ° C for 12,000 °C. After centrifugation at rpm for 20 min, the supernatant was removed; the pellet was resuspended in 200 ⁇ l 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 A 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, and 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.
  • 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 Ala-(D)Trp-Pro-Tyr-Val-Thr-Leu-NH 2 , wherein the second tryptophan is in the D configuration, represented by LRH7-G1, and the short peptide is Shanghai Qiang Yao Biological Company Synthetic.
  • Example 2 GPR1 antagonist polypeptide LRH7-G1 can effectively alleviate the inhibitory effect of chemerin on cAMP signaling pathway.
  • Cyclic adenosine monophosphate (cAMP) enzyme-linked immunosorbent assay
  • sample concentration is determined by BCA method
  • cAMP cyclic adenosine monophosphate
  • 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-G1 (3 ⁇ M, 0.3 ⁇ M, 0.03 ⁇ M) 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-G1 did not significantly inhibit the effect of chemerin on cAMP concentration. Similarly, LRH7-G1 did not increase cAMP concentration in the LRH7-G1 short peptide alone group.
  • LRH7-G1 can specifically inhibit the decrease of cAMP concentration by chemerin by acting with GPR1.
  • Example 3 The GPR1 antagonist polypeptide LRH7-G1 can effectively inhibit the chelation of calcium (Ca 2+ ) induced by chemerin.
  • Reagent configuration Mix probenecid into 1mL buffer solution, configure to a concentration of 250nM probenecid, shake well, add fluorescent reagents to prepare for use;
  • 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. .
  • chemerin can significantly reduce the calcium ion (Ca 2+) concentrations, inhibition chemerin activation of calcium (Ca 2+) signaling pathway stream.
  • chemerin has no activation effect on calcium (Ca 2+ ) flow signaling pathway
  • LRH7-G1 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-G1 short peptide can specifically inhibit the chemerin/GPR1 signaling pathway to reduce calcium (Ca 2+ ) concentration.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Pharmacology & Pharmacy (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Engineering & Computer Science (AREA)
  • Diabetes (AREA)
  • Endocrinology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biochemistry (AREA)
  • Rheumatology (AREA)
  • Epidemiology (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Pain & Pain Management (AREA)
  • Reproductive Health (AREA)
  • Immunology (AREA)
  • Emergency Medicine (AREA)
  • Hematology (AREA)
  • Obesity (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

本发明公开了一种GPR1拮抗多肽及其衍生物与应用，所述多肽序列如SEQ ID NO.1-5所示。该拮抗多肽在体外能结合GPR1，通过阻断chemerin与GPR1的结合而促进cAMP浓度增加、抑制chemerin引起的钙内流，为高表达GPR1受体疾病如乳腺癌等提供有效的治疗小分子药物，能够在医学与生物学领域得到广泛的应用。

Description

一种GPR1拮抗多肽及其衍生物与应用 技术领域
本发明涉及生物技术和生物医药领域，具体而言，本发明是雌性生殖疾病靶点GPR1受体拮抗多肽LRH7-G1及其衍生物与应用。
背景技术
乳腺癌(Breast cancer)是仅次于肺癌的全球第2高死亡率的致命性癌症，它持续性破坏着全世界数以百万计的妇女及其家庭的生命健康。据世界卫生组织国际癌症研究中心最新统计，2012年全球女性乳腺癌新发病例达167万，占全部女性恶性肿瘤发病的22.9％；46万女性因乳腺癌死亡，占所有女性恶性肿瘤死亡的13.7％，占所有女性死亡的1.7％，大约有4个患癌妇女中就有1个患有乳腺癌。中国女性乳腺癌年龄标化发病率为21.6/10万，居女性癌症发病的第1位；死亡率为5.7/10万，居女性癌症死亡的第6位。4％～6％乳腺癌诊断时即为转移性乳腺癌，而接受辅助治疗的早期患者30％～40％可发展为转移性乳腺癌，患者5年生存率约20％。而手术、放疗和化疗等传统治疗方式，作用的特异性和专一性不佳，不可避免的会对正常细胞和组织产生杀伤性作用，给患者带来极大的副作用。因此，寻找高效特异性的靶向药物，将极大提高乳腺癌的治疗效果！
临床研究发现，早期乳腺癌往往不具备典型的症状和体征，不易引起重视，常通过体检或乳腺癌筛查才能发现。虽然乳腺癌的早期诊断可以为乳腺癌的治疗赢得宝贵的时间，可以极大提高乳腺癌的治疗效果，但是我国目前进行早期乳腺癌诊断的人员比例很少。
乳腺癌术后转移至淋巴，是乳腺癌常见的一种转移症状。一般地说，乳房外侧病变向腋窝淋巴结转移的机会占2/3，向胸骨旁转移的机会为1/3。在乳房内侧和中央部的肿瘤，向内侧和外侧乳腺癌淋巴转移的机会各占1/2，乳腺癌容易发生肺转移和淋巴转移。肿瘤微环境对肿瘤的形成至关重要，决定了肿瘤的发生和发展。最先是正常的乳腺干细胞发生致癌突变转化为乳腺癌干细胞，产生预后不良的肿瘤，然后这些具有转移性的不良预后的肿瘤，在基质纤维母细胞的影响下，一部分乳腺癌干细胞发生转移，转移到脑部、肺部、肝脏以及骨髓。
有研究表明，趋化因子受体CXCR4在乳腺癌组织中呈现异常高表达，其配体基质细胞衍生因子－1(CXCL12)也高表达于乳腺癌转移部位，如骨髓、淋巴结、肺部以及肝脏中，CXCR4/CXCL12信号系统在乳腺癌的归巢向远处其他器官转移中发挥着重要的作用，因此也成为了乳腺癌骨转移潜在的治疗靶标，其中CXCR4拮抗多肽—CTCE-9908，已经进入临床前试验阶段，在乳腺癌动物模型中能够很好的抑制乳腺癌的骨转移。因此，趋化因子在乳腺癌发生发展和转移中发挥着重要的作用，以趋化因子为靶标治疗乳腺癌的靶向药物具有广阔的市场前景以及应用价值。
乳腺癌起源于乳腺各级别导管和腺泡上皮，由腺上皮增生到不典型增生而逐步发展为原位癌、早期浸润癌至浸润性癌。不同级别的导管发生的癌变，其组织类型常常不同。乳腺癌中95％以上是恶性上皮性肿瘤，乳腺肉瘤十分少见。组织病理学上最常见的两种侵润性乳腺癌分别为：侵润性导管癌(也被称为“普通型，NOS”)和小叶癌。这两种乳腺癌是由前侵润性导管和小叶瘤形成的。侵润性导管癌和小叶癌以及其它类型的乳腺癌的不同之处主要是由细胞之间的形态差异引起的。
乳腺癌分子靶向治疗是指针对乳腺癌发生、发展有关的信号通路及其癌基因相关表达产物进行治疗。受恶性肿瘤发病率居高不下的影响，全球抗肿瘤药物市场呈现持续增长之势，2010年抗肿瘤类药物的销售额达459.7亿美元，占世界药物销售额的5.8％，居世界第一，比2009年增长8％。其中，分子靶向抗肿瘤药物销售额为311.9亿美元，增长率达到23％。1997年美国食品和药物管理局(Food and Drug Administration，FDA)批准抗人表皮生长因子受体2(HER-2)的分子靶向治疗药物—曲妥珠单克隆抗体的应用，开始了乳腺癌分子靶向治疗的新时代。据全球畅销药数据统计，2015年全球乳腺癌药物市场规模已超过100亿美元，其中销售额最高的是曲妥珠单抗(Trastuzumab)，销售额为67.9亿美元，较同期增长4.2％。许多研究发现，表皮生长因子受体(EGFR)是调控乳腺癌发生和发展的关键分子，通过激活下游Ras/Raf/MEK/ERK和PI3K/AKT信号通路，EGFR促进了乳腺癌的增殖、迁移和侵袭[39,40]；另外，表皮生长因子受体2(HER-2)高表达于乳腺癌细胞，通过形成同源二聚体或异源二聚体，激活下游PI3K/Akt和Ras/MAPK信号通路促进乳腺癌增殖、血管生成、迁移和侵袭。目前，针对乳腺癌治疗的分子靶向药物主要有：针对表皮生 长因子受体-2治疗的曲妥珠单抗；针对PI3K/AKT/mTOR通路中的抑制剂依维莫司；针对抗血管生成药物的贝伐珠单抗；针对BRCA1/2突变的iniparib；以及针对CDK4/6抑制剂palbociclib。这些靶向药物的问世及应用为乳腺癌患者带来了新的曙光，使乳腺癌的治疗变得更加精确，极大地减轻了传统治疗带来的损伤副作用！
趋化因子及其受体在乳腺癌的发展过程中，不但与肿瘤微环境的建立及肿瘤转移密切相关，还可以介导肿瘤细胞的恶化，以及促进肿瘤细胞的生长及增殖。研究指出，CXCL8、CXCL12、CCL2、CCL5、CCL18等趋化因子具有促乳腺癌作用，而另外一些趋化因子如XCL1、CXCL9、CXCL10、CXCL14、CCL16、CCL19等则可能有抗乳腺癌作用。肿瘤相关成纤维细胞(CAFs)和癌细胞可以活化并分泌大量CCL2，促进肿瘤干细胞(CSC)的自我更新，进而强化肿瘤细胞对化、放疗的抵抗性并促进病灶的转移。CXCR4在乳腺癌中的阳性率高达60％，CXCL12/CXCR4作用网络在包括乳腺癌在内的多种实体瘤的生长、转移中发挥重要作用，阻断这一生物学轴可抑制血管生成和肿瘤扩散，增加肿瘤细胞对化疗、放疗的敏感性。在4T1小鼠乳腺癌模型，用携带CXCR4拮抗剂的溶瘤病毒治疗可抑制血管生成和肿瘤转移。TNBC中CXCR4的表达(71％)高于HER-2阳性乳腺癌(44％)和Luminal型乳腺癌(37％)，CXCR4+TNBC患者肿瘤大，易发生肝、肺、脑转移，预后差。趋化因子及其受体拮抗剂或抑制剂具有广阔的市场前景。有统计表明，约30％的小分子药物是以G蛋白偶联受体为靶向的[63]。CXCR4多肽类拮抗剂CTCE-9908已被证实可抑制小鼠骨肉瘤和黑色素瘤的肺转移。CXCR1/CXCR2拮抗剂—Repertaxin在动物模型中可抑制肿瘤的生长和转移，已用于中晚期乳腺癌患者临床试验。因此，研究趋化因子在肿瘤发展中的作用，寻找趋化因子治疗乳腺癌的新靶点，不仅对阐释肿瘤转移有着重要的意义，而且有广阔的药物市场前景。
肥胖与乳腺癌的相关性研究最早见于20世纪60年代，病例对照研究最早提出乳腺癌的发病倾向于绝经后肥胖及高血压病的女性。在动物实验研究中也发现，与对照组相比，肥胖的小鼠肿瘤生长速度明显增加。世界癌症研究基金会和美国癌症研究所以流行病学证据进行回顾性分析，动物模型的研究结果证明超重和肥胖可显著增加患乳腺癌的危险性。Meta分析显示，患乳腺癌的危险性随BMI增加而增加，BMI＞30kg/m2的患者患乳腺癌的风险是正常人的1.3～ 2倍，肥胖可显著增加女性患乳腺癌的风险。
肥胖促进乳腺癌发生、发展的可能机制包括以下几个方面:(1)雌激素。肥胖会增加雌激素的合成，降低血清雌二醇与性激素结合球蛋白(SHBG)水平，血清雌激素过量，竞争靶器官上的雌激素受体，引起下丘脑-垂体-卵巢性腺轴功能紊乱，打破乳腺组织增生与复旧的平衡，促进乳腺癌细胞的异常增殖与转化；(2)慢性炎症。肥胖患者脂肪组织中活化的巨噬细胞可分泌大量促炎介质，引起慢性炎症反应，大量炎性因子和体内各种细胞为肿瘤形成了肿瘤微环境，从而促进乳腺癌细胞的发生、发展。当血液中TG浓度过高时，乳腺血管脂质沉积导致慢性炎症的发生，炎症细胞分泌的炎症因子与间质成纤维细胞、淋巴细胞、巨噬细胞、血管及淋巴管网络和细胞外基质形成肿瘤微环境，干扰DNA的复制和转录，导致恶性肿瘤发生；(3)胰岛素和胰岛素样生长因子-1(IGF-1)。肥胖易出现胰岛素抵抗，使胰岛素及IGF-1的水平升高，有助于癌细胞的增殖和血管再生。一项来自瑞士数据库的研究收集了2005年接受胰岛素治疗的115 000例患者的资料，发现在接下来的2年时间里单用甘精胰岛素治疗的患者乳腺癌发病风险明显较使用其他胰岛素类似物高(RR＝1.99)。(4)脂肪细胞因子。肥胖会影响脂肪细胞因子的产生，降低脂联素，升高瘦素、PAI-1等的水平，促进细胞增殖、抗凋亡及加速血管生成，从而促进癌细胞的增殖及生长。脂联素不仅可以抑制胰岛素与雌激素协同促进的有丝分裂作用，还可以通过促进Bax和p53促凋亡基因的表达，降低癌细胞的转移和浸润能力。瘦素能够通过激活JAK/STAT3，MAPK-ERK1/2或者PI3K通路，从而促进乳腺癌的生长；另外，瘦素还能通过诱导血管生成素的表达促进肿瘤相关血管的生成，并且瘦素还能诱导人表皮生长因子受体2ErbB-2)的转录，并且参与三阴性乳腺癌细胞中胰岛素样生长受体1(IGF-1)的反应，激活表皮生长因子受体(EGFR)从而促进细胞的侵袭与转移。肥胖是绝经后乳腺癌的危险因素，肥胖与乳腺癌发生、发展的关系密切且复杂，大量研究表明肥胖对乳腺癌的发生、发展、诊断、肿瘤特点及预后均可产生一定的负向调控。因此，通过对肥胖、脂肪组织以及其分泌的脂肪因子与乳腺癌关系的进一步探索和研究，可为今后制定降低乳腺癌发病率和改善疾病预后的相应措施提供非常重要的参考依据。
脂肪因子Chemerin
脂肪因子Chemerin也称为他扎罗汀诱导基因2(TIG2)或视黄醇受体反应 蛋白2。在1997年被Nagpal等克隆发现后，在2003年，Wittamer等在卵巢癌继发的腹水中通过反相高压液相层析分离得到其活性蛋白。Chemerin蛋白前体全长163个氨基酸，具有6个半胱氨酸残基，形成3个二硫键，去掉N端信号肽和C端的几个氨基酸，才具有生物活性，其血液中的chemerin活化形式含有134个氨基酸(约16kDa)，在结构上属于杀菌肽/半胱氨酸蛋白酶抑制剂家族，当发生炎症反应时，在几种蛋白酶作用下chemerin能迅速转变成活化形式。Chemerin在人体的多种组织广泛表达，主要表达于白色脂肪组织、胎盘和肝脏，与3个受体相结合：①G蛋白偶联受体CMKLR1；②G蛋白偶联受体GPR1；③CCRL-2。Chemerin结合GPR1后，一方面可以使GPR1阳性细胞内的钙离子释放，抑制cAMP的聚集，使MAP激酶磷酸化；另外一方面对细胞内钙离子内流具有促进作用。Chemerin一方面作为趋化因子，趋化树突状细胞和巨噬细胞，在免疫和适应性免疫之间起桥梁作用，趋化天然杀伤细胞到达炎症部位参与炎症反应；另一方面作为新的脂肪因子，由脂肪组织分泌产生，调节脂肪细胞的分化、脂解，可促进脂肪细胞内胰岛素信号传导途径等生物学效应。Chemerin在肥胖和代谢综合征的病理生理机制中占重要的地位。
GPR1受体
GPR1是CMKLR1最接近的同系物，与CMKLR1共享超过40％的序列同一性。它是一种在真核细胞中起关键作用的G蛋白偶联受体，通常对葡萄糖刺激，脂质积累以及将营养信号传递给cAMP途径。许多信号转导通过真核生物中的G蛋白→偶联受体(GPCR)介导。G蛋白β偶联受体存在于一些真核生物中，包括酵母，双鞭毛虫和动物。GPR1最初是在人类中发现的G蛋白偶联受体(GPCR)，其通过体外实验被鉴定为chemerin的受体。虽然没有报道Chemerin与GPR1结合的生理功能，但据报道，GPR1在鼠动物棕色脂肪组织，白色脂肪组织和骨骼肌中高度表达，并且GPR1主要在白色脂肪组织的血管细胞中表达。在喂食高脂肪饮食的GPR1敲除小鼠中，发现其葡萄糖不耐受比WT小鼠更严重。此外，在丙酮酸耐受试验中，GPR1敲除小鼠能够抑制葡萄糖α刺激的胰岛素水平升高，引起血糖升高。这些结果表明，GPR1是Chemerin的活性受体，可以调节肥胖期间葡萄糖体内的平衡。
使用Tango生物测定，测量在增加Chemerin剂量治疗后CMKLR1和GPR1 在HTLA细胞中的活性。发现Chemerin以相似的功效激活CMKLR1和GPR1，达到效果为265.3±182.2-和185.6±26.5-倍变化的最大反应(Emax)。此外，Chemerin激活GPR1比激活CMKLR1(EC50,54.6±30.7nM)具有显著更高的效力(EC50,18.2±2.9nM)，这证明Chemerin激活了GPR1，同时GPR1也是高度敏感的Chemerin受体。
GPR1表达细胞系通过弱Ca 2+动员和ERK1/2激活对chemerin作出反应，但其由于激动剂结合而有效地内化。因此，GPR1可能作为chemerin的诱饵受体，而迄今为止，在主要细胞内或体内尚未有对GPR1介导的活性的描述。并且GPR1在中枢神经系统，骨骼肌，皮肤和脂肪组织中表达，可能调节chemerin的活性。
G蛋白偶联受体超家族(GPCRs)在胞外信号向胞内转导过程中起到重要的作用，调控着细胞运动、生长和基因转录—这三个癌症生物学中至关重要的因素。过去的十几年中，介导的信号通路已经被证明是原癌基因信号的关键调控者，并且是很好的药物靶点，目前市面上出售的药物60％都是针对该受体设计的。而GPR1作为GPCRs成员之一，目前仍然没有任何药物是直接针对GPR1来治疗临床癌症的。因此，以GPR1为靶标，筛选抗癌的新型多肽药物和人源化抗体具有重要的社会意义和广阔的经济市场。
噬菌体展示技术(Phage Display Technology)是一项特异性多肽或蛋白的筛选技术，此技术可将目的基因编码的多肽以融合蛋白的形式展示于噬菌体表面，被展示的多肽或蛋白可以保持相对独立的空间结构和生物活性，使大量随机多肽与其DNA编码序列之间建立了直接联系，使得各种靶分子(如抗体、酶和细胞表面受体等)的多肽配体通过体外亲和淘选程序得以快速鉴定。自从问噬菌体展示技术世以来，因其高库容、高效方便以及灵活筛选等优势，噬菌体抗体库技术在近几十年内进展非常迅速，在生命科学的许多领域得到广泛应用，尤其在肿瘤诊断和肿瘤抗体药物制备等领域正越来越受到的重视。目前多肽药物的主要作用靶点是G蛋白偶联受体(GPCRs)，在临床研究中有39％多肽药物靶向GPCRs。多肽药物具有许多优点：⑴成药性高于一般化学药物，其生物活性高，特异性强；⑵毒性反应相对较弱，在体内不易产生蓄积；⑶与其他药物的相互作用比较少，与体内受体的亲和性比较好。人源化单克隆抗体具有灵敏度高、特 异性强、高效、血清交叉反应少或无、制备成本低等优点。利用噬菌体展示技术筛选多肽药物和人源化抗体，可以实现基因型和表型的统一，是一种制备抗体高效、实用的技术方法，该技术利用表达蛋白与配体的亲和力，将所需要的蛋白或多肽筛选出来，广泛用于疾病标志物的识别筛选和抗体药物的筛选和制备之中。
发明内容
为了克服现有技术的不足和缺点，本发明的首要目的在于提供一种由噬菌体展示库筛选得到的GPR1拮抗多肽，该拮抗多肽与Chemerin受体GPR1具有特异性高亲和力，能够通过抑制Chemerin与GPR1的结合来阻断Chemerin/GPR1的信号通路，其在高表达GPR1受体的疾病的靶向治疗方面具有巨大的应用价值。
本发明的另一目的在于提供上述GPR1受体拮抗多肽的衍生物，该衍生物也能够与GPR1受体具有特异性高亲和力，且特异性竞争Chemerin与GPR1的结合位点，能够抑制Chemerin结合GPR1。
本发明的再一目的在于提供上述GPR1拮抗多肽及其衍生物的应用。
为了实现上述任务，本发明采取如下的技术解决方案：
GPR1拮抗多肽，其特征在于，其氨基酸序列为:Ala-(D)Trp-Pro-Tyr-Val-Thr-Leu-NH 2SEQ ID No.1。即LRH7-G1。
上述GPR1拮抗多肽的筛选方法，该方法利用噬菌体随机肽库，首先采用GPR1质粒转染293T细胞，获得永久高表达GPR1的稳定细胞系，以野生型293T细胞为对照吸附细胞，进行5轮全细胞消减筛选，随机挑取50个阳性噬菌体扩增，提取克隆单链DNA测序。分析多肽的氨基酸序列的基本特征，多肽同源性比较，检索出现频率高的多肽基序。BLAST检索蛋白质数据库，检测多肽基序同源性较高的蛋白质，发现含有大量该多肽的生物物种，及可能结合的细胞表面受体和配体，有利于后续大量提取和纯化获得多肽。
亲水性分析表明，LRH7-G1为疏水性多肽；
高效液相色谱法(HPLC)和质谱(MS)检测LRH7-G1合成的纯度达到了99.85％。
本发明另一个方面提供了本发明所述的GPR1受体拮抗多肽的衍生物为GPR1受体拮抗多肽氨基酸侧链基团上、GPR1受体拮抗多肽片段的氨基端或羧基端进行常规修饰得到的产物，或者为GPR1受体拮抗多肽上连接用于多肽 或蛋白检测或纯化的标签所得到的产物；
优选地，所述的常规修饰为氨基化、酰胺化、羟基化、羧基化、羰基化、烷基化、乙酰化、磷酸化、硫酸化、酯化、糖基化、环化、生物素化、荧光基团修饰、聚乙二醇PEG修饰或固定化修饰等；
优选地，所述的标签为His6、GST、EGFP、MBP、Nus、HA、IgG、FLAG、c-Myc或ProfinityeXact。
本发明再一个方面提供了本发明所述的GPR1拮抗多肽的生物活性片段或类似物，其如SEQ ID No.2-5所示：
AWPYVTL        SEQ ID No.2
AWPYVTL-NH 2    SEQ ID No.3
WPYVTL         SEQ ID No.4
WPYVTL-NH 2     SEQ ID No.5
本发明还提供了GPR1受体拮抗多肽的生物活性片段或类似物的衍生物，所述GPR1受体拮抗多肽的生物活性片段或类似物的衍生物的氨基酸侧链基团上、氨基端或羧基端进行常规修饰得到的产物，或者为GPR1受体拮抗多肽的生物活性片段或类似物上连接用于多肽或蛋白检测或纯化的标签所得到的产物；
优选地，所述的常规修饰为氨基化、酰胺化、羟基化、羧基化、羰基化、烷基化、乙酰化、磷酸化、硫酸化、酯化、糖基化、环化、生物素化、荧光基团修饰、聚乙二醇PEG修饰或固定化修饰等；
优选地，所述的标签为His6、GST、EGFP、MBP、Nus、HA、IgG、FLAG、c-Myc或ProfinityeXact。
在本发明的技术方案中，所述的GPR1拮抗多肽及其衍生物可以应用于制备预防和/或治疗雌性生殖疾病方面的药物，其存在形式有：①含有6个氨基酸；②由7个氨基酸组成。所述的GPR1拮抗多肽的衍生物为GPR1拮抗多肽氨基酸侧链基团上、GPR1拮抗多肽片段的氨基端或羧基端进行常规修饰得到的产物，或者为GPR1拮抗多肽上连接用于多肽或蛋白检测或纯化的标签所得到的产物；所述的常规修饰优选为氨基化、酰胺化、羟基化、羧基化、羰基化、烷基化、乙酰化、磷酸化、酯化、糖基化、环化、生物素化、荧光基团修饰、聚乙二醇PEG修饰或固定化修饰等；所述的标签优选为His 6、GST、EGFP、MBP、Nus、HA、IgG、FLAG、c-Myc或ProfinityeXact等；
所述的GPR1拮抗多肽及其衍生物可以来源于哺乳类动物或者鸟类，例如灵长类动物(人类)；啮齿类动物，包括小鼠，大鼠，仓鼠，兔，马，牛，犬类，猫等。
所述的GPR1拮抗多肽的衍生物优选为：上述GPR1拮抗多肽第二个氨基酸残基为D构型色氨酸，末端进行酰胺化修饰，即为Ala-(D)Trp-Pro-Tyr-Val-Thr-Leu-NH 2。
所述的GPR1拮抗多肽及其衍生物的获得，采用现有技术中的公知方法进行，既可以用多肽自动合成仪进行化学合成；通过将短肽序列推导出核苷酸序列，然后克隆到载体中进行生物合成；也可以从现有存在的生物体内进行大量提取和纯化。
具体来说，GPR1拮抗多肽LRH7-G1及其衍生物包括以下天然存在于生物中的多肽：
SEQ ID No.2     AWPYVTL(真菌)
SEQ ID No.3     AWPYVTL-NH 2(真菌)
SEQ ID No.4     WPYVTL(昆虫、鸟、贝类、真菌、细菌、豆类、猫、熊、犬)
SEQ ID No.5     WPYVTL-NH 2(昆虫、鸟、贝类、真菌、细菌、豆类、猫、熊、犬)。
本发明再一个方面提供了一种多聚核苷酸，其编码SEQ ID No.1-5任一项所述多肽。
本发明再一个方面提供了一种载体，其包含了本发明所述的核苷酸，可以通过基因技术手段与启动子序列链接。
本发明再一个方面提供了一种宿主细胞，其转染了本发明所述的载体。
本发明再一个方面提供了本发明所述的GPR1受体拮抗多肽、GPR1受体拮抗多肽的衍生物、GPR1受体拮抗多肽的生物活性片段或类似物及其衍生物在制备治疗趋化素-GPR1介导疾病的药物中的用途。
本发明再一个方面提供了本发明所述的GPR1受体拮抗多肽、GPR1受体拮抗多肽的衍生物、GPR1受体拮抗多肽的生物活性片段或类似物及其衍生物在治疗趋化素GPR1介导疾病中的用途。
在本发明的技术方案中，所述趋化素-GPR1介导疾病选自乳腺癌、脂肪肝、 糖尿病、炎症反应、多囊卵巢综合症。
本发明再一个方面提供了本发明所述的GPR1受体拮抗多肽、GPR1受体拮抗多肽的衍生物、GPR1受体拮抗多肽的生物活性片段或类似物及其衍生物在制备抑制chemerin造成的cAMP浓度的降低的药物中的用途。
本发明再一个方面提供了本发明所述的GPR1受体拮抗多肽、GPR1受体拮抗多肽的衍生物、GPR1受体拮抗多肽的生物活性片段或类似物及其衍生物在抑制chemerin造成的cAMP浓度的降低中的用途。
本发明再一个方面提供了本发明所述的GPR1受体拮抗多肽、GPR1受体拮抗多肽的衍生物、GPR1受体拮抗多肽的生物活性片段或类似物及其衍生物在制备抑制chemerin引起的钙(Ca 2+)内流作用的药物中的用途。
本发明再一个方面提供了本发明所述的GPR1受体拮抗多肽、GPR1受体拮抗多肽的衍生物、GPR1受体拮抗多肽的生物活性片段或类似物及其衍生物在抑制chemerin引起的钙(Ca 2+)内流作用中的用途。
本发明再一个方面提供了本发明所述的GPR1受体拮抗多肽、GPR1受体拮抗多肽的衍生物、GPR1受体拮抗多肽的生物活性片段或类似物及其衍生物在制备抑制chemerin引起的细胞趋化作用的药物中的用途。
本发明再一个方面提供了本发明所述的GPR1受体拮抗多肽、GPR1受体拮抗多肽的衍生物、GPR1受体拮抗多肽的生物活性片段或类似物及其衍生物在抑制chemerin引起的细胞趋化作用中的用途。
本发明再一个方面提供了本发明所述的GPR1受体拮抗多肽、GPR1受体拮抗多肽的衍生物、GPR1受体拮抗多肽的生物活性片段或类似物及其衍生物在制备治疗乳腺癌、脂肪肝、糖尿病、炎症反应、多囊卵巢综合症的药物中的用途。
本发明再一个方面提供了本发明所述的GPR1受体拮抗多肽、GPR1受体拮抗多肽的衍生物、GPR1受体拮抗多肽的生物活性片段或类似物及其衍生物在治疗乳腺癌、脂肪肝、糖尿病、炎症反应、多囊卵巢综合症中的用途。
本发明再一个方面提供了一种药物组合物，其中包含本发明所述的GPR1受体拮抗多肽、GPR1受体拮抗多肽的衍生物、GPR1受体拮抗多肽的生物活性片段或类似物及其衍生物中的一种或多种作为活性成分。
所述的药物组合物可以含有一种或者是多种药学上可以接受的载体；
所述药学上可以接受的载体优选为稀释剂、赋形剂、填充剂、粘合剂、湿润剂、崩解剂、吸收促进剂、吸附载体、表面活性剂或润滑剂等；
所述的药物组合物可以进一步制成片剂、粒剂、胶囊、口服液或注射剂等多种形式，各种剂型的药物可以按照药学领域的常规方法制备；
一种预防和/或治疗高表达GPR1受体的疾病方面的药物，包含GPR1受体拮抗多肽、GPR1受体拮抗多肽的衍生物、GPR1受体拮抗多肽的生物活性片段或类似物及其衍生物中的至少一种。
一种预防和/或治疗雌性生殖疾病或肿瘤方面的药物，包含本发明所述的GPR1受体拮抗多肽、GPR1受体拮抗多肽的衍生物、GPR1受体拮抗多肽的生物活性片段或类似物及其衍生物中的至少一种。
在本发明的一具体实验中，本发明利用所获得的GPR1拮抗多肽LRH7-G1可以有效的缓解chemerin对cAMP信号通路的抑制作用。其中，GPR1拮抗多肽LRH7-G1衍生物(SEQ ID No.1～5)具有相同作用。
在本发明的另一具体实验中，利用GPR1拮抗多肽LRH7-G1可以有效的抑制chemerin引起的钙(Ca 2+)内流作用。其中，GPR1拮抗多肽LRH7-G1衍生物(SEQ ID No.1～5)具有相同作用。
本发明相对于现有技术具有如下的优点及效果:
(1)本发明提供了一种GPR1拮抗多肽LRH7-G1及其衍生物(SEQ ID No.1～5)，所述的拮抗多肽及其衍生物能够专一性与GPR1结合，并特异性竞争Chemerin与GPR1结合位点，能够抑制Chemerin/GPR1信号通路。可以作为Chemerin/GPR1结合位点的生物类多肽药物，可用于制备预防和/或治疗高表达GPR1受体的疾病方面的药物，例如：乳腺癌、脂肪肝、糖尿病以及炎症反应、多囊卵巢综合症。能够在医学与生物学领域得到广泛的应用，并产生巨大的社会与经济效益。
附图说明
图1：LRH7-G1高效液相(HPLC)检测图。
图2：LRH7-G1质谱(MS)检测图。
图3：GPR1，chemerin以及LRH7-G1多肽疏水轮廓比较分析。其中：A：GPR1疏水轮廓图；B：chemerin疏水轮廓图；C：LRH7-G1多肽疏水轮廓图； D：GPR1，chemerin以及LRH7-G1多肽疏水轮廓比较图。GPR1轮廓为红色，chemerin轮廓为蓝色，LRH7-G1轮廓为绿色；
图4：LRH7-G1缓解chemerin对cAMP信号通路的抑制作用。其中，A，LRH7-G1与Forsklin和Chemerin在293T野生型细胞中的作用；B，LRH7-G1单独在293T GPR1 +/+中的作用；C，LRH7-G1与Forsklin和Chemerin在293T GPR1 +/+中的作用；
图5：LRH7-G1抑制chemerin引起的钙(Ca 2+)内流作用。
具体实施方式
为了更清楚地理解本发明，现参照下列实施例及附图进一步描述本发明。实施例仅用于解释而不以任何方式限制本发明。实施例中，各原始试剂材料均可商购获得，未注明具体条件的实验方法为所属领域熟知的常规方法和常规条件，或按照仪器制造商所建议的条件。
实施例1：进行GPR1拮抗多肽LRH7-G1的淘选、扩增、纯化、测序及合成。
本实施例主要是为了筛选获得与GPR1特异性结合的阳性噬菌体，再通过将阳性噬菌体扩增、纯化，提取噬菌体单链DNA(ssDNA)进行测序，将所获得的序列分析对比，最后合成高纯度的拮抗多肽LRH7-G1。
具体如下：
1.建立永久高表达GPR1的293T细胞系：293T-GPR1 +/+/LRH
①选取生长旺盛的可发光的人293T细胞，在转染前一天以5×10 5个/孔，接种于6孔板中，培养至第二日后，细胞融合度为60％；
②第二日进行转染，以6孔板的一个培养孔为单位，用200μL的opti-MEM培养基稀释3μg质粒，另以200μL的opti-MEM培养基稀释6μL脂质体Lipofectamine2000，分别轻轻混匀后，室温放置5分钟；
③将两管稀释液轻轻混合，室温静置20分钟后，向混合后的稀释液中轻轻加入600μL的opti-MEM培养基；
④将待转染的细胞用PBS轻轻漂洗一次，然后将混合好的稀释液轻轻加入培养孔中，置于二氧化碳培养箱中培养；
⑤培养4～6小时后弃尽转染所用培养基，向孔中加入3mL完全培养基；
⑥48小时后选用含有1μg/mL嘌呤霉素(puromycin)的培养基进行筛选；待细胞不再出现死亡后即得到稳定表达GPR1的293T细胞系。
⑦用TRIzol提取总RNA，定量2μg RNA进行逆转录(逆转录试剂盒，购于Promega公司)，并用特异引物序列进行qPCR。
所用特异引物序列为Hu-GPR1引物序列：
Fw 5’-AATGCCATCGTCATTTGGTT-3’(SEQ ID No.6)
Rv 5’-CAACTGGGCAGTGAAGGAAT-3’(SEQ ID No.7)
⑧与转染了pSM2c-Hu-scramble RNA的做比较，检测GPR1的高表达水平，并命名为：293T-GPR1 +/+/LRH，即可以用于阳性噬菌体筛选。
2.进行GPR1拮抗多肽的淘选、扩增、纯化、测序及合成。
①ER2738宿主菌液的制备：无菌技术操作，先取200μl LB-Tet液体培养基于1.5ml灭菌离心管中，再从E.coli ER2738的甘油冻存物中取0.2μl菌液与之充分混匀，全部吸取涂布于LB-Tet平板上，标记平板，室温放置3min，然后置于37℃恒温培养箱倒置过夜培养。次日观察，长出克隆后用封口膜封口，4℃避光保存备用。用灭菌枪头以无菌技术挑取单菌落，放入已预先加有3ml LB-Tet液体培养基的10ml灭菌离心管中，标记后于恒温摇床37℃，300rpm/min振荡培养过夜。次日，细菌扩增液于4℃储存备用。取10ml灭菌离心管，无菌操作加入3ml LB-Tet液体培养基，取30μl过夜培养的细菌接种其中，恒温摇床37℃，300rpm/min振荡培养2～3h，细菌处于指数生长期，肉眼观察呈雾状(OD 600～0.5)。
②GPR1拮抗肽的淘选：将高表达GPR1细胞按10 5个/培养皿接种于预先包被多聚赖氨酸的60×15mm 2培养皿中，常规培养至细胞密度80％～90％时，用于淘洗(同时用不表达GPR1的细胞系作为空白对照)每轮洗脱液先取1μl测滴度，剩下的将其加入到20ml LB培养液中扩增，再纯化、最后再测扩增后的滴度，扩增物于4℃短期保存，并取相等数量级用于下一轮淘选，剩下 的扩增物用50％的甘油于-20℃保存。
③测定噬菌体的滴度:取4支灭菌的10ml离心管，每个噬菌体稀释度准备1个灭菌离心管，微波炉熔化顶层琼脂(Agarose Top)，每管加入3ml顶层琼脂，45℃水浴备用。每个噬菌体稀释度准备1块LB/IPTG/Xgal平板，37℃恒温培养箱预热备用。将OD 600～0.5的E.coli ER2738大肠杆菌按照噬菌体稀释度200μl/管分装，4℃保存备用。取4支灭菌的1.5ml离心管，分别盛有100μl、90μl、90μl、90μl LB-Tet培养基，将待测噬菌体吸取1μl入100μl LB-Tet培养基中，按10倍梯度稀释，分别标记为10 -1、10 -2、10 -3、10 -4，每个稀释度轻轻振荡混匀，瞬间离心。取10μl待滴定的各稀释度的噬菌体与200μl E.coli ER2738混合，轻轻振荡混匀，瞬间离心，室温孵育5min。将混合菌液迅速加入顶层琼脂中，快速振荡混匀，立即倒入预热的LB/IPTG/Xgal平板，将其均匀展平，室温冷却5min，37℃恒温培养箱中，倒置平板培养过夜。
④洗脱噬菌体的扩增及纯化：取250ml的锥形瓶，按1:100比例，将过夜培养的ER2738宿主菌液加入至20ml LB液体培养基中，37℃，250rpm剧烈振荡培养2h；然后将待扩增的噬菌体液加入到锥形瓶中，37℃，250rpm剧烈振荡培养4.5h；将培养物转入50ml离心管中，4℃ 10,000rpm离心10min。上清液转入另一干净离心管中，4℃ 10,000rpm再次离心10min；取上清的80％转入另一干净离心管中，加入1/4体积的PEG/NaCl，颠倒混匀后于4℃沉淀过夜；第二天，将沉淀于4℃,12,000rpm离心20min。用干净枪头小心吸取上清液，再4℃ 12,000rpm离心1min，去掉残留上清液；然后用1ml TBS重悬沉淀物，轻轻吹打100次。然后将悬液转入2ml离心管中，4℃10,000rpm离心5min除去残余细胞；将上清加入1/4体积的PEG/NaCl后，于冰上孵育60min再次沉淀；取出离心管，4℃ 12,000rpm离心20min，去掉上清；用200μl TBS重悬沉淀，4℃ 10,000rpm离心1min。上清转入另一离心管中。4℃短期保存，也可以用50％的甘油于-20℃长期保存。单克隆噬菌体的扩增，包括⑴按1:100比例，将过夜培养的ER2738宿主菌液加入至2mL LB液体培养基中，37℃，250rpm剧烈振荡培养2h；用灭菌牙签，从第四轮滴度平板中选 取少于100个噬菌斑的平板，挑取分隔良好的蓝色噬菌斑，加入到培养管中，37℃ 250r/min剧烈震荡培养4.5h；然后将培养物转入到新鲜离心管中，4℃10,000rpm离心30sec。上清转入以新鲜管中，再同样离心一次；将上清的80％转入新鲜离心管中，4℃贮存，也可以用50％的甘油于-20℃长期保存。
⑤琼脂糖凝胶电泳鉴定M13噬菌体ssDNA：将凝胶成形模具水平放置，将选好的梳子放好，梳子底部与模具之间留1mm空间；称取DNA电泳用琼脂糖1g放入250ml的三角烧瓶中，加入100ml 1×TAE缓冲液，混匀后，将烧瓶置于微波炉中，加热煮沸，直至琼脂糖完全溶解；关闭电磁炉，取出三角烧瓶，将其置室温下冷却至室温(手握烧瓶可以耐受)，再加入溴化乙锭5μl，混匀后，即将凝胶溶液倒入胶板铺板。本实验所用制胶板约需胶液100ml；室温下待凝胶完全凝固，需时约30分钟，拔出梳齿，将胶板放入电泳槽中；在电泳槽加入1×TAE缓冲液，以高出凝胶表面2mm为宜；将样品用Loading buffer稀释以后加入胶板中，注意加样器吸头应恰好置于凝胶点样孔中，不可刺穿凝胶，也要防止将样品溢出孔外；接通电源，调节电压至50伏，电泳90min后，将凝胶板取出，在紫外灯下观察结果。
⑥ssDNA测序及序列分析：将提取的M13噬菌体ssDNA送到上海英潍捷基生物技术有限公司进行DNA测序。测序以后用Bioedit软件进行序列分析。通过分析结果可知，样品序列为Ala-(D)Trp-Pro-Tyr-Val-Thr-Leu-NH 2，其中第二位色氨酸为D构型，以LRH7-G1表示，最后短肽由上海强耀生物公司合成。
图1高效液相色谱法(HPLC)及图2质谱(MS)检测LRH7-G1多肽的纯度为99.85％，并且分子量大小与预测值一致。图3的疏水性轮廓分析，证明LRH7-G1多肽与chemerin有一定的相似性，相似度达到0.0122699(PAM250)。
实施例2GPR1拮抗多肽LRH7-G1可以有效的缓解chemerin对cAMP信号通路的抑制作用。
(1)环磷酸腺苷(cAMP)酶联免疫吸附实验：
①细胞铺板：将野生型293T细胞和高表达GPR1的293T细胞(293T GPR1 +/+)，分别以5ⅹ10 5个/孔接种于6孔细胞培养板中，每孔培养基体积为1mL，放置于培养箱中培养24h后，饥饿过夜，加入不同浓度梯度(3μM，0.3μM，0.03μM)的LRH7-G1多肽，Fosklin(25μM)以及chemerin(30nM)作用6h；
②样品制备：每孔加入300μL的细胞裂解液，4℃放置20分钟，用细胞刮刀刮取收集细胞，上下颠倒混匀后，12,000rpm离心10分钟，收集上清；
③样品浓度测定：样品浓度通过BCA法测定；
④环磷酸腺苷(cAMP)酶联免疫吸附测试：
a,准备所需试剂，每个样品准备3个复孔；
b,将50μL的样品或标准品加入包被有抗体的96孔板中；再在每孔中加入25μL cAMP过氧化物酶示踪剂缀合物；
c,每孔加入50μL Anti-cAMP抗体，室温，摇床上缓慢孵育30分钟；
d,使用洗脱液清洗5次，每孔加入100μL化学反光剂，室温孵育5分钟；
e,酶标仪读板，记录发光值。
图4显示，chemerin在浓度为30nM的时候可以降低细胞cAMP浓度，但是在高表达GPR1的293T细胞(293T GPR1 +/+)中加入不同浓度的LRH7-G1(3μM，0.3μM，0.03μM)作用后，可以使cAMP浓度显著增高。而在野生型293T细胞中，不表达GPR1受体，因此LRH7-G1对chemerin降低cAMP浓度的作用无明显抑制作用。同样的，在LRH7-G1短肽单独作用组中，LRH7-G1也不能使cAMP浓度增高。综上所述，可以推断出，LRH7-G1可以特异性的通过与GPR1作用来进一步抑制chemerin对cAMP浓度的降低作用。*P<0.05,**P<0.01,***P<0.001与Forskolin+chemerin组相比较。
实施例3GPR1拮抗多肽LRH7-G1可以有效的抑制chemerin引起的钙(Ca 2+)内流作用。
①细胞铺板：将野生型293T细胞和高表达GPR1的293T细胞(293T GPR1 +/+)，分别以5ⅹ10 3个/孔接种于96孔细胞培养板中，每孔培养基体积为200μL，放置于培养箱中培养24h后，饥饿过夜；
②试剂配置：将丙磺舒融入1mL缓冲溶液中，配置成浓度为250nM的丙磺舒，摇匀后，加入荧光试剂中准备待用；
③去掉细胞培养基，加入不同浓度梯度(30μM，3μM，0.3μM，0.03μM，0.003μM)的LRH7-G1多肽以及chemerin(0.3nM)作用30分钟，然后每孔加入100μL上述荧光试剂；
④37℃放置30分钟，然后室温放置30分钟；
⑤在激发光494nm和发射光516nm处测量荧光吸光值。
图5显示，在高表达GPR1的293T细胞(293T GPR1 +/+)中，chemerin在作用浓度为0.3nM时，可以促进钙(Ca 2+)流信号通路，增高钙离子(Ca 2+)浓度。而在加入不同浓度的LRH7-G1短肽后，可以显著降低钙离子(Ca 2+)浓度，抑制chemerin对钙(Ca 2+)流信号通路的激活。但是在野生型不表达GPR1的293T细胞中，chemerin对钙(Ca 2+)流信号通路无激活作用，LRH7-G1对钙(Ca 2+)流信号通路也无作用效果，通过该实验可以推断出，chemerin可以通过与受体GPR1结合来激活钙(Ca 2+)流信号通路，而LRH7-G1短肽可以特异性的抑制chemerin/GPR1信号通路来降低钙离子(Ca 2+)浓度。*P<0.05,**P<0.01,***P<0.001与chemerin组相比较。

Claims (11)

  1. 一种GPR1拮抗多肽，其特征在于，其氨基酸残基序列为Ala-(D)Trp-Pro-Tyr-Val-Thr-Leu-NH 2SEQ ID No.1。
  2. 一种GPR1拮抗多肽的生物活性片段或类似物，其如SEQ ID No.2-5所示：
    Figure PCTCN2017117256-appb-100001
  3. 权利要求1所述的GPR1拮抗多肽或权利要求2所述的GPR1拮抗多肽的生物活性片段或类似物的衍生物，其特征在于：
    所述的衍生物为GPR1拮抗多肽或者其生物活性片段活类似物的氨基酸侧链基团上、氨基端或羧基端进行常规修饰得到的产物，或者为连接用于多肽或蛋白检测或纯化的标签所得到的产物；
    优选地，所述的常规修饰为氨基化、酰胺化、羟基化、羧基化、羰基化、烷基化、乙酰化、磷酸化、酯化、糖基化、环化、生物素化、荧光基团修饰、聚乙二醇PEG修饰或固定化修饰；所述的标签为His 6、GST、EGFP、MBP、Nus、HA、IgG、FLAG、c-Myc或ProfinityeXact。
  4. 一种多聚核苷酸，其可以编码SEQ ID No.1-5任一项所述多肽。
  5. 一种载体，其包含了权利要求4中所述的任一条多聚核苷酸，并通过基因技术手段与启动子序列链接。
  6. 一种宿主细胞，其转染了权利要求5中所述的载体。
  7. 权利要求1所述的GPR1受体拮抗多肽、权利要求2所述的生物活性片段或类似物以及权利要求3所述的衍生物在治疗趋化素-GPR1介导疾病中的用途；
    优选地，所述趋化素-GPR1介导疾病选自乳腺癌、脂肪肝、糖尿病、炎症 反应、多囊卵巢综合症。
  8. 权利要求1所述的GPR1受体拮抗多肽、权利要求2所述的生物活性片段或类似物以及权利要求3所述的衍生物在制备抑制chemerin造成的cAMP浓度的降低的药物中的用途。
  9. 权利要求1所述的GPR1受体拮抗多肽、权利要求2所述的生物活性片段或类似物以及权利要求3所述的衍生物在制备抑制chemerin引起的钙(Ca 2+)内流作用的药物中的用途。
  10. 权利要求1所述的GPR1受体拮抗多肽、权利要求2所述的生物活性片段或类似物以及权利要求3所述的衍生物在制备抑制chemerin引起的细胞趋化作用的药物中的用途。
  11. 一种药物组合物，其中包含权利要求1所述的GPR1受体拮抗多肽、权利要求2所述的生物活性片段或类似物以及权利要求3所述的衍生物中的一种或多种，作为活性成分；
    优选地，所述的药物组合物可以含有一种或者是多种药学上可以接受的载体；
    所述药学上可以接受的载体优选为稀释剂、赋形剂、填充剂、粘合剂、湿润剂、崩解剂、吸收促进剂、吸附载体、表面活性剂或润滑剂等；
    所述的药物组合物可以进一步制成片剂、粒剂、胶囊、口服液或注射剂多种形式，各种剂型的药物可以按照药学领域的常规方法制备。
PCT/CN2017/117256 2017-12-19 2017-12-19 一种gpr1拮抗多肽及其衍生物与应用 WO2019119273A1 (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/CN2017/117256 WO2019119273A1 (zh) 2017-12-19 2017-12-19 一种gpr1拮抗多肽及其衍生物与应用

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2017/117256 WO2019119273A1 (zh) 2017-12-19 2017-12-19 一种gpr1拮抗多肽及其衍生物与应用

Publications (1)

Publication Number Publication Date
WO2019119273A1 true WO2019119273A1 (zh) 2019-06-27

Family

ID=66994293

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2017/117256 WO2019119273A1 (zh) 2017-12-19 2017-12-19 一种gpr1拮抗多肽及其衍生物与应用

Country Status (1)

Country Link
WO (1) WO2019119273A1 (zh)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040235749A1 (en) * 1999-09-15 2004-11-25 Sylvain Chemtob G-protein coupled receptor antagonists
WO2007149807A1 (en) * 2006-06-20 2007-12-27 Invitrogen Corporation Methods for identifying modifiers of gpr1 activity
CN107188928A (zh) * 2017-06-30 2017-09-22 河南大学淮河医院 四种具有结合hiv‑1病毒ccr5共受体活性的多肽及其应用

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040235749A1 (en) * 1999-09-15 2004-11-25 Sylvain Chemtob G-protein coupled receptor antagonists
WO2007149807A1 (en) * 2006-06-20 2007-12-27 Invitrogen Corporation Methods for identifying modifiers of gpr1 activity
CN107188928A (zh) * 2017-06-30 2017-09-22 河南大学淮河医院 四种具有结合hiv‑1病毒ccr5共受体活性的多肽及其应用

Similar Documents

Publication Publication Date Title
CN109942670B (zh) 一种gpr1拮抗多肽及其衍生物与应用
CN109942673B (zh) 一种gpr1拮抗多肽及其衍生物与应用
WO2019119275A1 (zh) 一种gpr1拮抗多肽及其衍生物与应用
WO2020168850A1 (zh) Ube3a泛素化PP2A激活因子PTPA在治疗天使综合症和孤独症中的应用
CN109942681B (zh) 一种gpr1拮抗多肽及其衍生物与应用
CN109942683B (zh) 一种cmklr1拮抗多肽及其衍生物与应用
WO2019119273A1 (zh) 一种gpr1拮抗多肽及其衍生物与应用
WO2019119272A1 (zh) 一种gpr1拮抗多肽及其衍生物与应用
Read et al. Cloning and characterization of chicken stromal cell derived factor-1
CN109942669B (zh) 一种gpr1拮抗多肽及其衍生物与应用
CN109942674B (zh) 一种gpr1拮抗多肽及其衍生物与应用
CN109942671B (zh) 一种gpr1拮抗多肽及其衍生物与应用
CN109942672B (zh) 一种gpr1拮抗多肽及其衍生物与应用
CN109942679B (zh) 一种gpr1拮抗多肽及其衍生物与应用
CN109942680B (zh) 一种gpr1拮抗多肽及其衍生物与应用
CN109942676B (zh) 一种cmklr1拮抗多肽及其衍生物与应用
CN109942684B (zh) 一种cmklr1拮抗多肽及其衍生物与应用
CN109942682B (zh) 一种cmklr1拮抗多肽及其衍生物与应用
US7638489B2 (en) Modulation of angiogenesis by Bartonella henselae
WO2019119307A1 (zh) 一种cmklr1拮抗多肽及其衍生物与应用
WO2019119303A1 (zh) 一种cmklr1拮抗多肽及其衍生物与应用
WO2019119302A1 (zh) 一种cmklr1拮抗多肽及其衍生物与应用
CN109942675B (zh) 一种cmklr1拮抗多肽及其衍生物与应用
CN109942677B (zh) 一种cmklr1拮抗多肽及其衍生物与应用
KR100958943B1 (ko) 쥐알파12/쥐알파13 단백질 기능의 하향 조절제를 포함하는전환성장인자베타 1 (티쥐에프 베타 1) 관련 질병의 예방또는 치료용 약학 조성물

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 17935510

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

32PN Ep: public notification in the ep bulletin as address of the adressee cannot be established

Free format text: NOTING OF LOSS OF RIGHTS PURSUANT TO RULE 112(1) EPC (EPO FORM 1205A DATED 16/11/2020)

122 Ep: pct application non-entry in european phase

Ref document number: 17935510

Country of ref document: EP

Kind code of ref document: A1