WO2019119302A1 - Polypeptides antagoniste de cmklr1, dérivés et applications de ceux-ci - Google Patents

Polypeptides antagoniste de cmklr1, dérivés et applications de ceux-ci Download PDF

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WO2019119302A1
WO2019119302A1 PCT/CN2017/117504 CN2017117504W WO2019119302A1 WO 2019119302 A1 WO2019119302 A1 WO 2019119302A1 CN 2017117504 W CN2017117504 W CN 2017117504W WO 2019119302 A1 WO2019119302 A1 WO 2019119302A1
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cmklr1
seq
chemerin
antagonist polypeptide
cells
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PCT/CN2017/117504
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English (en)
Chinese (zh)
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张键
代小勇
陈杰
黄晨
肖天霞
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深圳先进技术研究院
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    • 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
    • A61K38/10—Peptides having 12 to 20 amino acids
    • 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
    • C07K11/00—Depsipeptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof

Definitions

  • the present invention relates to the fields of biotechnology and biomedicine, and in particular, the present invention relates to a female reproductive disease target CMKLR1 receptor antagonist polypeptide and a derivative thereof and use thereof.
  • ovarian cancer is a malignant tumor that occurs in the ovary, 90% to 95% of which are primary ovarian cancers, and 5% to 10% of the primary cancers in other parts are transferred to the ovaries.
  • ovarian cancer is a malignant tumor that occurs in the ovary, 90% to 95% of which are primary ovarian cancers, and 5% to 10% of the primary cancers in other parts are transferred to the ovaries.
  • ovarian cancer was found to have no obvious symptoms at an early stage. However, some studies have shown that ovarian cancer may show some clinical symptoms in the early stage, such as abnormal bloating, fullness, abdominal pain or back pain, lack of energy, etc., but these symptoms are often ignored by patients, often when diagnosed Metastasis has occurred, and ovarian cancer is often described as a "silent killer", which in turn leads to a poor prognosis for ovarian cancer.
  • ovarian cancer is often spread to distant organs by direct spread and basin and abdominal cavity.
  • the main route of ovarian cancer metastasis is intra-abdominal implantable metastasis.
  • the main part of metastasis is the omentum, and 80% of women with ovarian cancer have retinal metastasis.
  • the cancer cells on the greater omentum grow at a much faster rate than the original lesion.
  • the omentum is a peritoneum that connects the stomach to the transverse colon. It contains phagocytic cells and has important defense functions. It is also an adipose tissue containing a large number of lipid droplets.
  • adipose tissue is mainly adipose tissue
  • adipose tissue is the largest endocrine organ in the body, which can secrete adipokines, cytokines, etc.
  • the relationship between fat cells and ovarian cancer cells in adipose tissue and its mechanism still need more More evidence is stated.
  • leptin secreted by white adipose tissue promotes the development of breast cancer: on the one hand, leptin can promote the growth of breast cancer by activating JAK/STAT3, MAPK-ERK1/2 or PI3K pathways; Can also promote tumor-associated angiogenesis by inducing expression of angiopoietin, and leptin can also induce transcription of human epidermal growth factor receptor 2 (ErbB-2) and participate in insulin-like growth in triple-negative breast cancer cells.
  • the reaction of body 1 (IGF-1) activates the epidermal growth factor receptor (EGFR) to promote cell invasion and metastasis.
  • leptin can also promote the development of various cancers such as prostate cancer and thyroid cancer, and its expression level is positively correlated with tumorigenesis, but the level of leptin in pancreatic cancer is relatively low, and both. The relationship is not very clear. Studies have also found that adiponectin has an inhibitory effect on the development of tumors.
  • Adipose tissue affects ovarian cancer in obese women.
  • Studies have also shown that IL-6 and IL-8 secreted by the adipocytes of the omentum in the peritoneal cavity can promote the transfer of ovarian cancer cells to it.
  • Chemerin is a newly discovered adipocytokine, also known as chemerin, which plays a role in immune response, inflammatory response, adipocyte differentiation maturation, lipid metabolism, etc., and is associated with obesity and metabolic syndrome.
  • the chemerin gene also known as Tazarotene Induced Gene 2 (TIG2), was first cloned in 1997. Subsequently, in 2003, Wittamer et al. isolated in ascites secondary to ovarian cancer by reversed-phase high pressure liquid chromatography. Its active protein.
  • chemerin and its receptor CMKLR1 chemokine receptor-1
  • hGCs major human ovarian granulosa cells
  • KGN human ovarian granulosa-like tumor cells
  • 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.
  • CMKLR1 is highly expressed in macrophages, immature dendritic cells and white adipose tissue, and is the main receptor for Chemerin to play biological functions.
  • Chemerin can chemotactically express CMKLR1 dendritic cells and macrophages, in immunity and adaptation.
  • Sexual immunity plays a role as a bridge between immune response, inflammatory response, adipocyte differentiation and maturation, lipid metabolism, etc., and is associated with obesity and metabolic syndrome.
  • Chemerin binds to CMKLR1, it releases calcium ions in CMKLR1-positive cells, inhibits aggregation of cAMP, and phosphorylates MAP kinase.
  • Pretreatment with pertussis toxin can block intracellular signal transduction of CMKLR1, and intracellular signal transduction of CMKLR1 may be related to Gi family.
  • 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.
  • RvE1 was identified as 5S, 12R, 18R-hydroxy-6Z, 8E, 10E, 14Z, 16E-eicosapentaenoic.
  • the vasopressin compounds (Rvs) are a group produced by epoxidation of EPA or DHA in vascular endothelial cells. According to the different substrates required, it can be divided into D, E and other types, each type is divided into several categories.
  • RvE1 belongs to the E-group Rvs, and Resolvin E1 is another lipid ligand of CMKLR1, which can reduce the inflammatory response, reduce the migration of dendritic cells and reduce the production of interleukin 12 by binding to CMKLR1 and the leukotriene B4 receptor BLT1.
  • RvE1 can be synthesized by an aspirin-dependent pathway and a non-dependent aspirin pathway.
  • RvE1 should be stimulated to produce a local action and rapidly metabolize further by enzymes. In mammalian tissues, there are at least four separate pathways of RvE1 metabolism. RvE1 has a strong anti-inflammatory effect, which can alleviate leukocyte-mediated tissue damage and down-regulate inflammatory gene overexpression. RvE1 is an important regulator of neutrophils. In an inflammatory model, including yeast-induced peritonitis and 2,4,6-trinitrobenzene-induced colitis, a nanogram dose of RvE1 can significantly reduce neutrophil infiltration at the site of inflammation. In CMKLR1 transfected cells, RvE1 interacts with CMKLR1 to inhibit tumor necrosis factor- ⁇ -induced NF- ⁇ B activation.
  • RvE1 blocks DC migration and IL-12 production.
  • RvE1 can effectively inhibit lipopolysaccharide (LPS)-stimulated release of TNF- ⁇ , IL-6 and IL-23 from bone marrow-derived dendritic cells (BMDCs).
  • LPS lipopolysaccharide
  • BMDCs bone marrow-derived dendritic cells
  • RvE1 blocks excessive platelet aggregation under pathological conditions, but does not interfere with collagen-induced physiological coagulation.
  • RvE1 induces L-selectin shedding and down-regulates the expression of human granulocyte and monocyte surface molecule CD18.
  • RvE1 rapidly reduces the rolling of leukocyte testicular venous epithelial cells.
  • RvE1 selectively induces the expression of the anti-adhesion molecule CD55 in epithelial cells, thereby promoting neutrophil clearance dependent on epithelial cell surface CD55. It can prevent inflammatory cells from trans-epithelial and transendothelial migration; promote phagocytosis of apoptotic neutrophils by macrophages; down-regulate the secretion of interleukin-12 from dendritic cells; up-regulate the expression of CCR5 in T cells; regulate the expression of leukocyte adhesion molecules Reduce leukocyte rolling; selectively block adenosine and thromboxane receptor agonist U46619 to promote platelet aggregation. It has exhibited good preventive and therapeutic effects in various disease models such as rabbit periodontitis, mouse peritonitis, mouse retinopathy, and colitis.
  • CMKLR1 also known as ChemR23.
  • Chemerin is a natural ligand for CMKLR1 by reverse pharmacology.
  • the human CMKLR1 gene is located at 23.3 of the upper arm of chromosome 12 and consists of three exons and two introns.
  • High expression in the acquired immune system in which high expression is detected in macrophages, immature dendritic cells (immatureDCs), monocytes, neutrophils, and natural killer cells, but expression in mature DCs is decreased; Small plate cells, adipocytes, endothelial cells, oral epithelial cells, osteoclasts, vascular smooth muscle cells, myocytes, and sacral glioma cells (DBTRG-05MG) also express CMKLR1.
  • Chemerin/RvE1 binds to CMKLR1 and activates endocytosis, which promotes the release of intracellular calcium (Ca 2+ ) ions, phosphorylates extracellular signal-regulated kinase 1/2 (ERK1/2), and binds to G-protein coupled Source trimers to inhibit the accumulation of cyclic adenosine monophosphate (cAMP); up-regulate the PI3K/Akt signaling pathway, down-regulate nuclear factor kappa B (NF ⁇ B) signaling pathway: recruit immature dendritic cells and macrophages to the site of inflammation, thereby Achieve regulation of various metabolic processes, inflammatory responses and cancer.
  • cAMP cyclic adenosine monophosphate
  • Chemerin can induce endothelial cell proliferation and neovascular plexus formation after binding to CMKLR1 receptor on endothelial cell membrane. It promotes the growth and proliferation of tumor cells.
  • the expression level of Chemerin in most tumors is very low, and some are not even detected, but in the paracancerous tissues, and in the distal normal tissues, Chemerin expression levels are higher, such as: colon cancer, lung cancer (including non- Small cell lung cancer), liver cancer, melanoma, gastric cancer and ovarian cancer.
  • GPCRs G-protein coupled receptor superfamilies
  • PhageDisplay Technology is a screening technology for specific polypeptides or proteins.
  • 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 maintain a relatively independent spatial structure.
  • biological activity which establishes a direct link between a large number of random polypeptides and their DNA coding sequences, enabling rapid identification of polypeptide ligands of various target molecules (such as antibodies, enzymes and cell surface receptors) by in vitro affinity panning procedures.
  • Phage display technology has been widely used in the screening of tumor diagnostic markers and anti-tumor lead compounds, tumor-specific antibodies and targeted drug delivery of tumor drugs.
  • the primary object of the present invention is to provide a CMKLR1 antagonist polypeptide which is screened by a phage display library, which has a specific high affinity with the Chemerin/RvE1 receptor CMKLR1 and can inhibit Chemerin.
  • /RvE1 binds to CMKLR1 to block the signaling pathway of Chemerin/RvE1-CMKLR1, demonstrating that this peptide plays an important role in targeting ovarian cancer cell proliferation and promoting ovarian cancer cell apoptosis.
  • the treatment has great application value.
  • Another object of the present invention is to provide a derivative of the above CMKLR1 receptor antagonist polypeptide which is also capable of having a specific high affinity with the CMKLR1 receptor and which specifically competes with the binding site of Chemerin/RvE1 and CMKLR1, and is capable of inhibiting Chemerin /RvE1 combines with CMKLR1.
  • a further object of the present invention is to provide the use of the above CMKLR1 antagonist polypeptide and derivatives thereof.
  • CMKLR1 antagonist polypeptide characterized in that the amino acid sequence LRH12-C1 is: Asp-(D) Tyr-His-Asp-Pro-Ser-Leu-Pro-Thr-Leu-Arg-Lys -NH 2 (SEQ ID No. 1); wherein Tyr is in the D configuration.
  • CMKLR1 antagonist polypeptide adopts a phage random peptide library, firstly transfects 293T cells with CMKLR1 plasmid to obtain a stable cell line with permanent high expression of CMKLR1, 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 CMKLR1 antagonist polypeptide of the present invention is obtained by conventionally modifying the amino terminus or the carboxy terminus of the CMKLR1 antagonist polypeptide fragment on the amino acid side chain group of the CMKLR1 antagonist polypeptide. Or a product obtained by ligating a tag for detection or purification of a polypeptide or protein on a CMKLR1 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.
  • CMKLR1 antagonist polypeptide of the invention as set forth in SEQ ID No. 2-18:
  • X 1 , X 2 and X 3 are, in order, self-tryptophan, aspartic acid, threonine (W, N, T);
  • X 4 is selected from the group consisting of phenylalanine and leucine (F, L);
  • X 5 , X 6 and X 7 are valine, valine and alanine (V, V, A);
  • X 9 and X 11 are glutamic acid (E);
  • X 12 and X 13 are sequentially selected from asparagine and serine (N, S).
  • X 14 is selected from methionine or isoleucine (M, I);
  • X 16 is selected from serine or alanine (S, A);
  • X 18 is selected from the group consisting of glutamic acid or aspartic acid (E, N);
  • X 20 and X 21 are phenylalanine and valine in turn;
  • X 22 is selected from alanine or glycine (A, G)
  • X 23 is selected from valine or serine (V, S)
  • X 25 is selected from asparagine or alanine (N, A);
  • X 27 is selected from the group consisting of glutamic acid or leucine (E, L);
  • X 28 is arginine (R);
  • X 29 is alanine (A);
  • the invention also provides a derivative of a biologically active fragment or analog of a CMKLR1 antagonist polypeptide, wherein the amino acid side chain group, the amino terminus or the carboxy terminus of the derivative of the biologically active fragment or analog of the CMKLR1 antagonist polypeptide is conventionally modified.
  • the resulting product, or a biologically active fragment or analog of a CMKLR1 antagonist polypeptide is ligated to a product obtained for labeling or purification of a polypeptide or protein;
  • 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 CMKLR1 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 8 amino acids; 2 consists of 9 amino acids. Composition; 3 consists of 10 amino acids; 4 consists of 11 amino acids; 5 consists of 12 amino acids; 6 consists of 18 amino acids; 7 consists of 24 amino acids.
  • the derivative of the CMKLR1 antagonist polypeptide is a product obtained by conventional modification of the amino acid side chain group of the CMKLR1 antagonist polypeptide, the amino terminus or the carboxy terminus of the CMKLR1 antagonist polypeptide fragment, or the CMKLR1 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;
  • CMKLR1 antagonist polypeptide and its derivatives may be derived from mammals, birds, bacteria or nematodes, such as primates (humans); rodents, including mice, rats, hamsters, rabbits, horses, Cows, dogs, cats, etc.
  • the derivative of the CMKLR1 antagonist polypeptide is: the second amino acid residue of the above CMKLR1 antagonist polypeptide is D-configuration tyrosine, and the terminal is amidated, that is, Asp-(D) Tyr-His-Asp-Pro -Ser-Leu-Pro-Thr-Leu-Arg-Lys-NH 2 .
  • CMKLR1 antagonist polypeptide and the derivative thereof is carried out 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.
  • CMKLR1 antagonist polypeptide and its derivatives have the following sequences:
  • X 1 , X 2 and X 3 are tryptophan, aspartic acid, threonine (W, N, T);
  • X 4 is selected from the group consisting of phenylalanine and leucine (F, L);
  • X 5 , X 6 and X 7 are valine, valine and alanine (V, V, A);
  • X 9 and X 11 are both glutamic acid (E);
  • X 12 and X 13 are asparagine and serine (N, S) in this order.
  • X 14 is selected from methionine or isoleucine (M, I);
  • X 16 is selected from serine or alanine (S, A);
  • X 18 is selected from the group consisting of glutamic acid or aspartic acid (E, N);
  • X 20 and X 21 are phenylalanine and valine in turn;
  • X 22 is selected from alanine or glycine (A, G)
  • X 23 is selected from valine or serine (V, S)
  • X 25 is selected from asparagine or alanine (N, A);
  • X 27 is selected from the group consisting of glutamic acid or leucine (E, L);
  • X 28 is arginine (R);
  • X 29 is alanine (A).
  • CMKLR1 antagonist polypeptides and derivatives thereof also 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. 1-17.
  • 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.
  • CMKLR1 antagonist polypeptide a derivative of a CMKLR1 antagonist polypeptide, a biologically active fragment or the like of a CMKLR1 antagonist polypeptide, and a derivative thereof are provided for the preparation of a medicament for treating a chemerin/RvE1-CMKLR1 mediated disease.
  • CMKLR1 antagonist polypeptide a derivative of a CMKLR1 antagonist polypeptide, a biologically active fragment or analog of a CMKLR1 antagonist polypeptide, and a derivative thereof, for use in treating a chemerin/RvE1-CMKLR1 mediated disease.
  • the chemerin/RvE1-CMKLR1 mediated disease is selected from the group consisting of ovarian cancer, polycystic ovary syndrome, fatty liver, diabetes, and inflammatory response.
  • CMKLR1 antagonist polypeptide a derivative of a CMKLR1 antagonist polypeptide, a biologically active fragment or analog of the CMKLR1 antagonist polypeptide, and a derivative thereof according to the present invention are provided in the preparation of a medicament for inhibiting a decrease in cAMP concentration caused by chemerin. the use of.
  • CMKLR1 antagonist polypeptide a derivative of a CMKLR1 antagonist polypeptide, a biologically active fragment or analog of a CMKLR1 antagonist polypeptide, and a derivative thereof, for inhibiting a decrease in cAMP concentration by chemerin is provided.
  • the CMKLR1 antagonist polypeptide, the derivative of the CMKLR1 antagonist polypeptide, the biologically active fragment or the like of the CMKLR1 antagonist polypeptide and the derivative thereof are provided in the preparation of calcium (Ca 2+ ) induced by inhibition of chemerin. Use in flow-acting drugs.
  • CMKLR1 antagonist polypeptide a derivative of a CMKLR1 antagonist polypeptide, a biologically active fragment or the like of the CMKLR1 antagonist polypeptide, and a derivative thereof are provided for inhibiting chemerin-induced calcium (Ca 2+ ) influx. Use in action.
  • CMKLR1 antagonist polypeptide a derivative of a CMKLR1 antagonist polypeptide, a biologically active fragment or the like of a CMKLR1 antagonist polypeptide, and a derivative thereof are provided in the preparation of a medicament for inhibiting chemotaxis caused by chemerin. the use of.
  • CMKLR1 antagonist polypeptide a derivative of a CMKLR1 antagonist polypeptide, a biologically active fragment or analog of a CMKLR1 antagonist polypeptide, and a derivative thereof, for inhibiting chemerin-induced cell chemotaxis is provided.
  • CMKLR1 antagonist polypeptide a derivative of a CMKLR1 antagonist polypeptide, a biologically active fragment or analog of the CMKLR1 antagonist polypeptide, and a derivative thereof are provided for treating ovarian cancer, polycystic ovary syndrome, and fat.
  • CMKLR1 antagonist polypeptide a derivative of a CMKLR1 antagonist polypeptide, a biological active fragment or the like of the CMKLR1 antagonist polypeptide, and a derivative thereof are provided for treating ovarian cancer, polycystic ovary syndrome, and fatty liver. , diabetes, and the use of inflammatory reactions.
  • a pharmaceutical composition comprising the CMKLR1 antagonist polypeptide of the present invention, a derivative of a CMKLR1 antagonist polypeptide, a biologically active fragment or analog of a CMKLR1 antagonist polypeptide, and a derivative thereof, or A variety of as an active ingredient.
  • 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 or a lubricant.
  • the pharmaceutical composition may be further prepared into various forms such as tablets, granules, capsules, oral solutions or injections, and the medicaments of various dosage forms may be prepared according to a conventional method in the pharmaceutical field.
  • a medicament for preventing and/or treating a female reproductive disease comprising at least one of the above-described CMKLR1 antagonist polypeptide, a derivative of a CMKLR1 antagonist polypeptide, a biologically active fragment or analog of a CMKLR1 antagonist polypeptide, and a derivative thereof kind.
  • the present invention utilizes the obtained CMKLR1 antagonist polypeptide LRH12-C1 to effectively alleviate the inhibitory effect of chemerin on cAMP signaling pathway.
  • the CMKLR1 antagonist polypeptide LRH12-C1 derivative (SEQ ID No. 1 to 17) has the same effect.
  • the CMKLR1 antagonist polypeptide LRH12-C1 can effectively inhibit the chelation of calcium (Ca 2+ ) induced by chemerin.
  • the CMKLR1 antagonist polypeptide LRH12-C1 derivative (SEQ ID No. 1 to 17) has the same effect.
  • chemerin can act by chemotaxis of cells by binding to the CMKLR1 receptor.
  • the inventors used the Transwell assay to find that the CMKLR1 antagonist polypeptide LRH12-C1 significantly inhibits cell migration induced by chemerin.
  • the CMKLR1 antagonist polypeptide LRH12-C1 derivative (SEQ ID No. 1 to 17) has the same effect.
  • CMKLR1 antagonist polypeptide LRH12-C1 was found to significantly inhibit ovarian cancer cell SKOV- 3 proliferation.
  • the CMKLR1 antagonist polypeptide LRH12-C1 derivative (SEQ ID No. 1 to 17) has the same effect.
  • the inventors used flow cytometry to detect that the CMKLR1 antagonist polypeptide LRH12-C1 can block the SKOV-3 cycle of ovarian cancer cells and promote the apoptosis of SKOV-3.
  • the CMKLR1 antagonist polypeptide LRH12-C1 derivative (SEQ ID No. 1 to 17) has the same effect.
  • the present invention provides a CMKLR1 antagonist polypeptide and a derivative thereof (SEQ ID No. 1 to 17), which are capable of specifically binding to CMKLR1 and specifically competing for Chemerin/RvE1
  • the binding site to CMKLR1 inhibits the Chemerin/RvE1-CMKLR1 signaling pathway.
  • CMKLR1 antagonist polypeptide and its derivative (SEQ ID NO: 1-17) screened by the present invention can inhibit the proliferation of breast cancer cells and promote the apoptosis of breast cancer cells by blocking the binding of Chemerin/RvE1-CMKLR1.
  • a biological polypeptide drug which can be used as a Chemerin/RvE1-CMKLR1 binding site, and can be used for the preparation of a medicament for preventing and/or treating female reproductive diseases, such as ovarian cancer, polycystic ovary syndrome, fatty liver, diabetes, and inflammatory reaction. It can be widely used in the fields of medicine and biology, and it has enormous social and economic benefits.
  • Figure 1 LRH12-C1 high performance liquid chromatography (HPLC) detection chart.
  • Figure 2 LRH12-C1 mass spectrometry (MS) detection plot.
  • Figure 3 Comparative analysis of hydrophobic profiles of CMKLR1, chemerin, RvE1 and LRH12-C1 polypeptides.
  • A CMKLR1 hydrophobic profile
  • B chemerin hydrophobic profile
  • C RvE1 hydrophobic profile
  • D LRH12-C1 polypeptide hydrophobic profile
  • E CMKLR1, chemerin, RvE1 and LRH12-C1 polypeptide hydrophobic profile comparison.
  • the CMKLR1 is blue, the chemerin is green, the RvE1 is red, and the LRH12-C1 is purple.
  • Figure 4 LRH12-C1 attenuates the inhibitory effect of chemerin on the cAMP signaling pathway.
  • Figure 5 LRH12-C1 inhibits the influx of calcium (Ca 2+ ) induced by chemerin.
  • Figure 6 LRH12-C1 inhibits cell chemotaxis by chemerin.
  • Figure 7 LRH12-C1 inhibits the proliferation of ovarian cancer cell SKOV-3.
  • Figure 8 LRH12-C1 blocks the cell cycle progression of ovarian cancer cell SKOV-3.
  • A flow cytometry to detect cell cycle;
  • B cell cycle data statistics.
  • Figure 9 LRH12-C1 promotes apoptosis of ovarian cancer cell line SKOV-3.
  • Example 1 Panning, amplification, purification, sequencing and synthesis of CMKLR1 antagonist polypeptides.
  • This example is mainly for screening for positive phage which specifically binds to CMKLR1, 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; after the cells no longer died, a 293T cell line stably expressing CMKLR1 was obtained.
  • CMKLR1 +/+ /LRH 293T-CMKLR1 +/+ /LRH, which can be used for positive phage screening.
  • 1ER2738 host bacterial solution preparation aseptic technique, first take 200 ⁇ l LB-Tet liquid medium in 1.5ml sterile centrifuge tube, then take 0.2 ⁇ l bacterial solution from E.coliER2738 glycerol frozen stock and mix well, all Suction coating The plate was placed on an LB-Tet plate, and the plate was labeled, placed at room temperature for 3 min, and then placed in a 37 ° C incubator for inversion overnight culture. The next day of observation, the clone was grown and sealed with a parafilm, and stored at 4 ° C in the dark.
  • a single colony was picked by a sterile technique using a sterile tip, placed in a 10 ml sterilized centrifuge tube to which 3 ml of LB-Tet liquid medium was previously added, labeled, and shake-cultured at 37 ° C, 300 rpm/min overnight at a constant temperature shaker. The next day, the bacterial amplification solution was stored at 4 ° C until use. Take 10ml sterile centrifuge tube, add 3ml LB-Tet liquid medium aseptically, take 30 ⁇ l of overnight cultured bacteria to inoculate it, shake incubator at 37°C, shake at 300rpm/min for 2 ⁇ 3h, the bacteria are in exponential growth phase, visual observation It is foggy (OD600 ⁇ 0.5).
  • CMKLR1 antagonist peptide Panning of 2CMKLR1 antagonist peptide: high-expression CMKLR1 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 CMKLR1 as a blank control), take 1 ⁇ l of titer for each round of eluate, and add the remaining to 20 ml of LB medium for amplification, purification, and finally amplification.
  • the amplification was stored for a short period of time at 4 ° C and was taken on an equal order for the next round of panning, and the remaining amplification was stored at 50 ° C with glycerol at -20 ° C.
  • phage titer Take 4 sterile 10 ml centrifuge tubes, prepare a sterile centrifuge tube for each phage dilution, melt the top agar (Agarose Top) in a microwave oven, add 3 ml of top agar to each tube, and use a 45 ° C water bath for use.
  • One LB/IPTG/Xgal plate was prepared for each phage dilution, and preheated in a 37 °C incubator.
  • E. coli ER2738 E. coli with OD600-0.5 was dispensed according to phage dilution 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, centrifuged again at 10,000 rpm for 10 min at 4 ° C; 80% of the supernatant was transferred to another clean centrifuge tube, 1/4 volume of PEG/NaCl was added, and the mixture was inverted and mixed.
  • the pellet was precipitated overnight at 4 ° C; the next day, the pellet was centrifuged at 12,000 rpm for 20 min at 4 ° C.
  • the supernatant was carefully aspirated with a clean tip and centrifuged at 12,000 rpm for 1 min at 4 ° C to remove the residual supernatant; then the pellet was resuspended in 1 ml of TBS and gently pipetted 100 times.
  • the suspension was then transferred to a 2 ml centrifuge tube and centrifuged at 10,000 rpm for 5 min at 4 ° C for 5 min to remove residual cells; the supernatant was added to 1/4 volume of PEG/NaCl, and then incubated on ice for 60 min to precipitate again; the centrifuge tube was removed, 12,000 °C at 4 °C After centrifugation at rpm for 20 min, the supernatant was removed; the pellet was resuspended in 200 ⁇ 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 of LB liquid medium at a ratio of 1:100, vigorously shaking at 37 ° C, 250 rpm for 2 h; using a sterile toothpick, from the fourth round The plate with less than 100 plaques was selected from the plate, and the well-separated blue plaques were picked and added to the culture tube, and cultured at 37 ° C for 250 h/min for 4.5 h; The culture was then transferred to a fresh centrifuge tube and centrifuged at 10,000 rpm for 30 sec at 4 °C.
  • 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; after the sample is diluted with Loadingbuffer, it is added into 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 from overflowing. Outside the hole; turn on the power, adjust the voltage to 50 volts, after electrophoresis for 90 min, remove the gel plate and observe the results 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 Asp-(D)Tyr-His-Asp-Pro-Ser-Leu-Pro-Thr-Leu-Arg-Lys-NH 2 , wherein the second tyrosine is in the D configuration. Expressed as LRH12-C1, the final short peptide was synthesized by Shanghai Qiangyao Biotech Co., Ltd.
  • Example 2 The CMKLR1 antagonist polypeptide LRH12-C1 can effectively alleviate the inhibitory effect of chemerin on cAMP signaling pathway.
  • Cyclic adenosine monophosphate (cAMP) enzyme-linked immunosorbent assay
  • CMKLR1 +/+ wild type 293T cells and 293T cells (293TCMKLR1 +/+ ) with high expression of CMKLR1 were seeded at 5 ⁇ 10 5 cells/well in 6-well cell culture plates at a volume of 1 mL per well. After incubation for 24 h in the chamber, starvation overnight, adding different concentration gradients (3 ⁇ M, 0.3 ⁇ M, 0.03 ⁇ M) of LRH12-C1 polypeptide, Fosklin (25 ⁇ M) and chemerin (30 nM) for 6 h;
  • 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 after adding different concentrations of LRH12-C1 (3 ⁇ M, 0.3 ⁇ M, 0.03 ⁇ M) in 293T cells (293 TCMKLR1 +/+ ) with high expression of CMKLR1 , can make the cAMP concentration significantly increased.
  • CMKLR1 receptor was not expressed, so LRH12-C1 had no significant inhibitory effect on chemerin's effect on reducing cAMP concentration.
  • LRH12-C1 did not increase cAMP concentration in the LRH12-C1 short peptide alone group.
  • LRH12-C1 can specifically inhibit the decrease of cAMP concentration by chemerin by acting with CMKLR1.
  • Example 3 The CMKLR1 antagonist polypeptide LRH12-C1 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 (293TCMKLR1 +/+ ) with high expression of CMKLR1, chemerin can promote calcium (Ca 2+ ) flow signaling pathway and increase calcium ion (Ca 2+ ) concentration at a concentration of 0.3 nM. After the addition of different concentrations of peptide LRH12-C1, it 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
  • LRH12-C1 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 CMKLR1, while the LRH12-C1 short peptide can specifically inhibit the chemerin/CMKLR1 signaling pathway to reduce calcium (Ca 2+ ) concentration.
  • Example 4 Transwell assay detects that the CMKLR1 antagonist polypeptide LRH12-C1 inhibits chemerin-induced cellular chemotaxis.
  • Figure 6 shows that in L1.2 cells (L1.2CMKLR1 +/+ ) with high expression of CMKLR1, chemerin can significantly promote cell chemotaxis from the upper chamber to the lower chamber, while adding the same concentration of LRH12-C1 short peptide, It can significantly reduce the number of cell chemotaxis and inhibit the chemotaxis of chemerin to cells.
  • chemerin in wild type L1.2 cells that do not express CMKLR1, chemerin has no chemotactic effect on cells, and LRH12-C1 short peptide has no effect on cells.
  • chemerin can promote chemotactic regulation by binding to the receptor CMKLR1, and the LRH12-C1 short peptide can specifically block the chemerin/CMKLR1 signaling pathway to inhibit cell chemotaxis.
  • Example 5 LRH12-C1 significantly inhibited the proliferation of human ovarian cancer SKOV-3 cells.
  • Human ovarian cancer cell line SKOV-3 was inoculated into a 96-well cell culture plate at 5 ⁇ 10 3 cells/well, and the medium volume per well was 200 ⁇ L, cultured for 24 hours, and then starved overnight;
  • Example 6 LRH12-C1 blocked the cell cycle progression of human ovarian cancer SKOV-3.
  • Human ovarian cancer cell line SKOV-3 was inoculated into a 6-well cell culture plate at 5 ⁇ 10 5 cells/well, and the volume of the culture medium per well was 1 mL, cultured for 24 hours, and then starved overnight;
  • the red fluorescence was detected by a flow cytometer at an excitation wavelength of 488 nm, and the light scattering was detected. Analyze the cell results.
  • Figure 8 shows that by flow cytometry analysis, on the one hand, with the increase of the concentration of action, LRH12-C1 short peptide can significantly reduce the number of cells in human ovarian cancer cells SKOV-3G2/M phase, increase G0/G1 and S phase The number of cells blocked the normal progression of the cell cycle; on the other hand, with the prolongation of the action time, the LRH12-C1 short peptide also significantly decreased the number of cells in the G2/M phase, increased the number of G0/G1 and S phases, and blocked the normal progression of the cell cycle.
  • the LRH12-C1 short peptide inhibits the proliferation of human ovarian cancer cell line SKOV-3 by blocking the cell cycle.
  • Example 7 LRH12-C1 can promote apoptosis of human ovarian cancer SKOV-3 cells.
  • Human ovarian cancer cell line SKOV-3 was inoculated into a 6-well cell culture plate at 5 ⁇ 10 5 cells/well, and the volume of the culture medium per well was 1 mL, cultured for 24 hours, and then starved overnight;
  • Figure 9 shows that by flow cytometry analysis, on the one hand, with the increase of the concentration of action, LRH12-C1 short peptide can significantly promote the apoptosis of human ovarian cancer cell line SKOV-3, on the other hand, with the prolongation of the action time
  • the LRH12-C1 short peptide also significantly increased the number of apoptosis in human ovarian cancer cell line SKOV-3.
  • the LRH12-C1 short peptide can significantly promote the apoptosis of human ovarian cancer cell line SKOV-3.

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Abstract

La présente invention concerne des polypeptides antagonistes de CMKLR1 (SEQ ID No : 1-17), des dérivés et des applications de ceux-ci. Les dérivés des polypeptides sont des produits obtenus par modification classique sur des groupes de chaînes latérales d'acides aminés des polypeptides antagonistes de CMKLR1 et sur des extrémités amino ou des extrémités carboxy de fragments polypeptidiques antagonistes de CMKLR1, ou des produits obtenus par fixation aux polypeptides antagonistes de CMKLR1, d'étiquettes pour la détection ou la purification de polypeptides ou de protéines. Les polypeptides et les dérivés polypeptidiques peuvent se lier à CMKLR1 in vitro, et en bloquant la liaison de chémérine à CMKLR1, peuvent favoriser l'augmentation de la concentration d'AMPc, inhibent l'influx calcique provoqué par la chémérine, inhibent la chimiotaxie induite par la chémérine, et peuvent inhiber la prolifération de cellules cancéreuses ovariennes, bloquer le cycle cellulaire de cellules cancéreuses ovariennes et favoriser l'apoptose dans des cellules cancéreuses ovariennes.
PCT/CN2017/117504 2017-12-20 2017-12-20 Polypeptides antagoniste de cmklr1, dérivés et applications de ceux-ci WO2019119302A1 (fr)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013117581A1 (fr) * 2012-02-10 2013-08-15 Charite - Universitätsmedizin Berlin Variants métaboliquement stables de chémérine 9
CN104434888A (zh) * 2013-09-17 2015-03-25 深圳先进技术研究院 Cmklr1小分子拮抗剂在防治非酒精性脂肪肝及肝炎中的应用
CN106749527A (zh) * 2016-12-28 2017-05-31 南京农业大学 氯噻啉抗体特异性结合的噬菌体展示多肽及其用途
CN107429249A (zh) * 2015-12-29 2017-12-01 深圳先进技术研究院 雌性生殖疾病靶点cmklr1及其拮抗剂与相关应用

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013117581A1 (fr) * 2012-02-10 2013-08-15 Charite - Universitätsmedizin Berlin Variants métaboliquement stables de chémérine 9
CN104434888A (zh) * 2013-09-17 2015-03-25 深圳先进技术研究院 Cmklr1小分子拮抗剂在防治非酒精性脂肪肝及肝炎中的应用
CN107429249A (zh) * 2015-12-29 2017-12-01 深圳先进技术研究院 雌性生殖疾病靶点cmklr1及其拮抗剂与相关应用
CN106749527A (zh) * 2016-12-28 2017-05-31 南京农业大学 氯噻啉抗体特异性结合的噬菌体展示多肽及其用途

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