WO2021129894A1 - 针对胰高血糖素样肽-1受体、胰高血糖素受体、以及抑胃肽受体的三重激动剂 - Google Patents

针对胰高血糖素样肽-1受体、胰高血糖素受体、以及抑胃肽受体的三重激动剂 Download PDF

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WO2021129894A1
WO2021129894A1 PCT/CN2021/075738 CN2021075738W WO2021129894A1 WO 2021129894 A1 WO2021129894 A1 WO 2021129894A1 CN 2021075738 W CN2021075738 W CN 2021075738W WO 2021129894 A1 WO2021129894 A1 WO 2021129894A1
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receptor
triple agonist
glucagon
peptide
triple
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PCT/CN2021/075738
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French (fr)
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辛中帅
李一飞
文良柱
钟远广
陈慧梅
张明义
赵梅
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万新医药科技(苏州)有限公司
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Priority to US17/788,738 priority Critical patent/US20230067960A1/en
Priority to EP21735129.5A priority patent/EP4095149A4/en
Publication of WO2021129894A1 publication Critical patent/WO2021129894A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/001—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof by chemical synthesis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/605—Glucagons
    • 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
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • 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
    • A61P3/06—Antihyperlipidemics
    • 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
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/12—Antihypertensives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to the field of biology, in particular to triple agonists that are active on GLP1 receptors, GCG receptors and GIP receptors, and related uses.
  • Obesity is one of the main causes of diabetes [1] .
  • the progress from obesity to diabetes is generally: obesity ⁇ impaired glucose tolerance ⁇ type 2 diabetes ⁇ uncontrollable hyperglycemia ⁇ diabetes complications. Therefore, for the treatment of diabetes, lowering blood sugar is one aspect, and weight reduction is also a factor that must be considered.
  • GLP-1 Glucagon-like peptide-1
  • GLP-1 is a polypeptide hormone secreted by intestinal L cells. It acts on pancreatic ⁇ cells in a glucose-dependent manner to promote the biosynthesis and secretion of insulin, and the increase of insulin brings obvious hypoglycemic effect; at the same time, it stimulates the proliferation and differentiation of ⁇ cells, inhibits ⁇ cell apoptosis, and increases pancreatic ⁇ cells Quantity, inhibit the secretion of glucagon, suppress appetite and food intake, and delay the emptying of stomach contents [2] .
  • Glucagon is a hormone secreted by islet alpha cells. It is opposed to insulin and plays a role in increasing blood sugar. Glucagon has a strong effect of promoting glycogen decomposition and gluconeogenesis, which can significantly increase blood sugar; at the same time, it can activate lipase, promote lipolysis, and can strengthen fatty acid oxidation, which increases the production of ketone bodies [3] . Therefore, although GCG is contrary to the goal of lowering blood sugar, it has obvious effects of lowering fat and weight.
  • the gastric inhibitory peptide or glucose-dependent insulin releasing peptide (GIP) is a type of hormone produced by K cells in the small intestine mucosa, which can inhibit gastric acid secretion; inhibit pepsinogen secretion; stimulate insulin release; inhibit gastric peristalsis And emptying; stimulate the secretion of intestinal fluid; stimulate the secretion of glucagon [4] .
  • GIP glucose-dependent insulin releasing peptide
  • the receptors for these three types of hormones belong to the GPCR receptor family, and have similar protein structures and binding mechanisms, making it possible to design multiple agonists for two or three receptors.
  • the triple agonists for GLP1-R, GCGR and GIPR can simultaneously perform the functions of three types of hormones [5, 6] .
  • GLP-1 can lower blood sugar and suppress appetite;
  • GCG can break down fat, reduce weight, but increase blood sugar;
  • GIP mainly stimulates insulin secretion, but also has the activity of stimulating GCG secretion.
  • the three cooperate with each other to form a feedback mechanism based on blood glucose concentration, which can not only control blood sugar, but also break down fat and reduce weight.
  • triple agonists have significant advantages over simple GLP-1 analogs.
  • the purpose of the present invention is to provide triple agonists against glucagon-like peptide-1 receptor (GLP1-R), glucagon receptor (GCGR), and gastric inhibitory peptide receptor (GIPR).
  • GLP1-R glucagon-like peptide-1 receptor
  • GCGR glucagon receptor
  • GIPR gastric inhibitory peptide receptor
  • Another object of the present invention is to provide a gene encoding the triple agonist, a recombinant expression vector including the gene, and a transformant including the gene or the recombinant expression vector.
  • Another object of the present invention is to provide a method for the synthesis, separation and purification of the triple agonist.
  • Another object of the present invention is to provide a method for chemical modification of certain amino acid side chains in the triple agonist.
  • Another object of the present invention is to provide a separation and purification method for preparing the triple agonist containing chemical modification.
  • Another object of the present invention is to provide a composition containing the triple agonist.
  • Another object of the present invention is to provide a method for treating a target disease, the method comprising providing the triple agonist or a composition containing the triple agonist to a subject.
  • Another object of the present invention is to provide the use of the triple agonist or the composition containing the triple agonist for the preparation of medicines, that is, for diabetes, obesity, hyperlipidemia, high cholesterol, non-alcoholic Fatty liver injury, as well as arteriosclerosis, atherosclerosis and coronary heart disease caused by hypercholesterolemia and hyperlipidemia.
  • the present invention provides isolated polypeptides with agonistic activity against glucagon-like peptide-1 receptor (GLP1-R), glucagon receptor (GCGR), and gastric inhibitory peptide receptor (GIPR), that is, against Triple agonist for glucagon-like peptide-1 receptor, glucagon receptor, and gastricin receptor.
  • GLP1-R glucagon-like peptide-1 receptor
  • GCGR glucagon receptor
  • GIPR gastric inhibitory peptide receptor
  • the triple agonist of the present invention is based on the natural Exendin-4 amino acid sequence, and at least one position has an amino acid substitution, mutation or chemical modification.
  • the triple agonist of the present invention is an amino acid sequence comprising the following general formula 1:
  • Xaa1 is glycine (Gly, G) or amino isobutyric acid (Aib);
  • Xaa2 is alanine (Ala, A) or arginine (Arg, R) or leucine (Leu, L);
  • Xaa3 is alanine (Ala, A) or glutamic acid (Glu, E) or tyrosine (Tyr, Y);
  • Xaa4 is methionine (Met, M) or leucine (Leu, L) or glutamine (Gln, Q);
  • Xaa5 is lysine (Lys, K) or chemically modified lysine K-R1 or K-R2 with a lipophilic side chain;
  • Xaa6 is glutamic acid (Glu, E) or isoleucine (Ile, I);
  • Xaa7 is alanine (Ala, A) or valine (Val, V);
  • Xaa8 is isoleucine (Ile, I) or arginine (Arg, R);
  • Xaa9 is aspartic acid (Asp, D) or glutamic acid (Glu, E);
  • Xaa10 is serine (Ser, S) or amidated serine (S-NH 2 ).
  • Xaa5 in Formula 1 is an amino acid K-R1 with a chemically modified side chain, wherein the chemical structure of K-R1 is as follows:
  • Xaa5 in Formula 1 is an amino acid K-R2 with a chemically modified side chain, wherein the chemical structure of K-R2 is as follows:
  • the triple agonist of the present invention is selected from the amino acid sequence comprising any one of the numbers 1-14 in Table 1:
  • K-R1 and K-R2 are as described above.
  • the triple agonist of the present invention has at least one of the following three activities:
  • the triple agonist of the present invention has a higher R1 or R2 modification on the K residue. Long half-life in vivo.
  • the C-terminus of the triple agonist of the present invention is amidated.
  • Another aspect of the present invention is to provide a gene encoding the triple agonist, a recombinant expression vector including the gene, and a transformant including the gene or the recombinant expression vector.
  • Another aspect of the present invention is to provide a method for synthesizing and isolating the triple agonist.
  • Another aspect of the present invention is to provide a method for chemical modification of certain amino acid side chains in the triple agonist.
  • Another aspect of the present invention is to provide a method for preparing the triple agonist containing chemical modification.
  • Another aspect of the present invention is to provide a composition containing the triple agonist, the composition further comprising a pharmaceutically acceptable carrier.
  • the composition is a pharmaceutical composition.
  • the present invention also provides the medical use of the triple agonist or the composition containing the triple agonist.
  • the present invention also provides the use of the triple agonist or the composition containing the triple agonist in the preparation of drugs for the prevention and/or treatment of metabolic syndrome diseases.
  • the metabolic syndrome of the present invention includes but is not limited to diabetes, obesity, hypertension, dyslipidemia, hypercholesterolemia, non-alcoholic steatohepatitis, and due to hypercholesterolemia, Arteriosclerosis, atherosclerosis and coronary heart disease caused by hyperlipidemia.
  • Another aspect of the present invention is to provide a method for treating a target disease, the method comprising providing the triple agonist or a composition containing the triple agonist to a subject.
  • the triple agonist of the present invention has activity against glucagon-like peptide-1 receptor (GLP1-R), glucagon receptor (GCGR), and gastric inhibitory peptide receptor (GIPR). These activities have been It is proved to be the therapeutic target of the above-mentioned metabolic syndrome, so the triple agonist of the present invention can be applied to the development and clinical treatment of preventive or therapeutic drugs for the above-mentioned metabolic syndrome.
  • GLP1-R glucagon-like peptide-1 receptor
  • GCGR glucagon receptor
  • GIPR gastric inhibitory peptide receptor
  • Figure 1 Preparation chromatogram of No. 5 peptide sample
  • Figure 2 UPLC analysis chromatogram of sequence number 5 peptide sample
  • Figure 3 The mass spectrum analysis diagram of the peptide sample of No. 5;
  • Figure 4 Preparation chromatogram of sequence number 6 peptide sample
  • Figure 5 UPLC analysis chromatogram of sequence number 6 peptide sample
  • Figure 6 Mass spectrometry analysis diagram of sequence number 6 peptide sample
  • Figure 8 UPLC analysis chromatogram of peptide sample No. 11;
  • Figure 9 The mass spectrum analysis diagram of the peptide sample of serial number 11;
  • Figure 10 Preparation chromatogram of the peptide sample of No. 14;
  • Figure 11 UPLC analysis chromatogram of No. 14 peptide sample.
  • Figure 12 Mass spectrometry analysis chart of No. 14 polypeptide sample.
  • amino acids mentioned in the present invention are abbreviated as follows according to the IUPAC-IUB naming rules:
  • amino acid residues in the polypeptide of the present invention are preferably in the L configuration.
  • the "-NH 2 "part on the C-terminus of the sequence represents the amide group (-CONH 2 ) on the C-terminus.
  • the unnatural amino acid aminoisobutyric acid (Aib) is also used.
  • GLP-1 and GIP receptors Compared with natural ligands (natural glucagon, natural GLP-1 and natural GIP), triple agonists that have a significant level of activity on glucagon, GLP-1 and GIP receptors have significant effects on glucagon,
  • One or more of GLP-1 and GIP receptors, specifically two or more receptors, and more specifically all three receptors, may present 0.1% or more, 1% or more High, 2% or higher, 3% or higher, 4% or higher, 5% or higher, 6% or higher, 7% or higher, 8% or higher, 9% or higher, 10% or higher, 20% or higher, 30% or higher, 40% or higher, 50% or higher, 60% or higher, 70% or higher, 80% or higher, 90% Or higher, and 100% or higher in vitro activity, but not particularly limited thereto.
  • the method for measuring the in vitro activity of the triple agonist can refer to Example 4 of the present invention, but is not particularly limited thereto.
  • the triple agonist may be an agonist that has an increased half-life in vivo after R1 or R2 modification on the K residue relative to any of natural GLP-1, natural glucagon and natural GIP, but is not particularly limited Here.
  • polypeptides described herein are analogs of natural exendin selected from substitutions, modifications, and combinations of substitutions on at least one amino acid of the natural Exendin-4 sequence, but not Especially limited to this.
  • the substitution of amino acids may include both substitutions with amino acids and substitutions with non-natural compounds.
  • the exendin analog may be selected from positions 2, 3, 12, 13, 14, 16, 17, 19, 27, 28, and 39 in the amino acid sequence of natural exendin. 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more of the amino acids , 9 or more, 10 or more, or 11 amino acids substituted by other amino acids, but not particularly limited thereto.
  • the amino acid substituted in the above natural exendin can be selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine , Isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine and aminoisobutyric acid , But not particularly limited to this.
  • amino acids to be replaced in the above-mentioned natural exendin can also choose amino acids whose side chains are chemically modified by lipophilic groups, specifically the side chains of lysine (Lys, K) residues are conjugated with lipophilic substituents.
  • lysine (Lys, K) residues are conjugated with lipophilic substituents.
  • the chemical structures of the two lipophilic chemically modified lysines K-R1 and K-R2 are as follows (the structural formulas of K-R1 and K-R2 in the following examples are the same here):
  • the polypeptide according to the present invention may include all forms of the polypeptide itself, its salt (for example, its pharmaceutically acceptable salt), or its solvate.
  • the polypeptide may be in any pharmaceutically acceptable form.
  • the kind of salt is not particularly limited, but is preferably a salt that is safe and effective for a subject (for example, a mammal), but is not particularly limited thereto.
  • pharmaceutically acceptable refers to a substance that can be effectively used for the intended use within the scope determined by pharmaco-medica1 without causing excessive toxicity, irritation, allergic reaction, etc.
  • solvate refers to the complex formed between the polypeptide or salt thereof and solvent molecules according to the present invention.
  • the triple agonist may be an agonist including any one of the amino acid sequences selected from No. 114 in Table 1, and (essentially) an agonist consisting of any one of the amino acid sequences selected from No. 1-14 in Table 1, but Not limited to this.
  • the triple agonist described herein can significantly activate at least one of glucagon receptor, GLP-1 receptor, and GIP receptor, but is not particularly limited thereto. Specifically, the triple agonist can significantly activate the GLP-1 receptor, or additionally significantly activate the glucagon receptor and/or the GIP receptor, but is not particularly limited thereto.
  • Another aspect of the present invention provides a polynucleotide encoding the triple agonist, a recombinant expression vector containing the polynucleotide, and a transformant containing the polynucleotide or the recombinant expression vector.
  • the recombinant vector of the present invention is not particularly limited, as long as the vector can replicate in host cells, it can be constructed using any vector known in the art.
  • the host used in the present invention is not particularly limited as long as it can express the polynucleotide of the present invention. Examples of suitable hosts include such as Escherichia coli, Pichia pastoris, insect cells, and animal cells such as CHO cells and the like.
  • polypeptide of the present invention can be synthesized by a solid-state automatic polypeptide synthesizer, or can be produced by genetic engineering technology.
  • the triple agonist of the present invention can be prepared by standard synthetic methods, recombinant expression systems, or any other methods known in the art. Therefore, the triple agonist of the present invention can be synthesized by many methods, including, for example, the method described below:
  • the target triple agonist can be produced by genetic engineering methods, which includes preparing a fusion gene encoding a fusion protein containing a fusion partner and the triple agonist, and transforming it into a host cell , Express the fusion protein, and then use a protease or compound to cleave the triple agonist from the fusion protein and then separate it.
  • Another aspect of the present invention is to provide a method for chemical modification of certain amino acid side chains in the triple agonist.
  • another aspect of the present invention is to provide a method for preparing the triple agonist containing chemical modification.
  • the chemical modification of the amino acid side chain involved in the present invention specifically refers to the chemical modification of the lipophilic group of the side chain of lysine.
  • K-R1 and K-R2 respectively.
  • CN1271086C for the chemical synthesis method of the side chain of the lipophilic group of R2 and the method of connecting to the side chain of lysine by reaction.
  • compositions containing the isolated triple agonist may be a pharmaceutical composition, and more specifically, a pharmaceutical composition for preventing or treating metabolic syndrome.
  • metabolic syndrome refers to symptoms that occur individually or in combination due to various diseases that occur due to chronic metabolic disorders.
  • diseases belonging to metabolic syndrome may include glucose intolerance, diabetes, obesity, hypertension, dyslipidemia, hypercholesterolemia, non-alcoholic steatohepatitis, and caused by hypercholesterolemia and hyperlipidemia.
  • Atherosclerosis, atherosclerosis and coronary heart disease but not limited to this.
  • Example 1 Synthesis and preparation of triple agonists without side chain chemical modification
  • X represents the unnatural amino acid aminoisobutyric acid (Aib).
  • Fmoc-AA-Wang resin with different amino acids is used as the starting material, and the deprotection coupling is carried out according to the synthetic sequence.
  • VS-P VS-C series peptide sequence synthesis until the end, and dried to obtain peptide resin.
  • the amount of ice methyl tert-butyl ether was washed 5 times and centrifuged to obtain the crude polypeptide.
  • the precipitate is reconstituted with triethylamine phosphate buffer, filtered with a 0.45 micron filter membrane, and set aside.
  • X represents the unnatural amino acid aminoisobutyric acid (Aib); -NH 2 represents the C-terminus of the amidated polypeptide (-CONH 2 ); K-R1 and K-R2 represent lysine, respectively.
  • X represents the unnatural amino acid aminoisobutyric acid (Aib); -NH 2 represents the C-terminus of the amidated polypeptide (-CONH 2 ); K-R1 and K-R2 represent lysine, respectively
  • the two lipophilic groups of the side chain are chemically modified, and the specific chemical structures are described above.
  • the synthetic crude peptides are vacuum-dried and dissolved with dilute ammonium bicarbonate solution to prepare a sample solution of about 5mg/ml, filtered with a 0.45 micron filter membrane, and set aside.
  • Peptide purification Use an aqueous solution containing 1% acetic acid and 20% ethanol as phase A, and an aqueous solution containing 1% acetic acid and 80% ethanol as phase B.
  • the collected components are evaporated to remove the organic solvent, and then settled with a dilute acetic acid aqueous solution, centrifuged, the precipitate is collected, washed with water, reconstituted, filtered, lyophilized, weighed, and the yield is calculated to be ⁇ 70%. Freeze storage after aliquoting. Analyze the corresponding mass spectrometry data according to the molecular weight of the target peptide, and the theoretical value and the actual measured value are within plus or minus 1.
  • X represents the unnatural amino acid aminoisobutyric acid (Aib); -NH 2 represents the C-terminus of the amidated polypeptide (-CONH 2 ); K-R1 and K-R2 represent lysine, respectively.
  • X represents the unnatural amino acid aminoisobutyric acid (Aib); -NH 2 represents the C-terminus of the amidated polypeptide (-CONH 2 ); K-R1 and K-R2 represent lysine, respectively
  • the two lipophilic groups of the side chain are chemically modified, and the specific chemical structures are described above.
  • Rink Amide-MBHA resin is used as the starting material, and the deprotection coupling is carried out according to the synthetic sequence.
  • the synthetic crude peptides are vacuum-dried to prepare a sample solution of about 5mg/ml with purified water, adjust the pH to between 7.0-9.0 with dilute ammonia, and filter with a 0.45 micron filter membrane for use.
  • Peptide purification use 80 mM ammonium acetate aqueous solution as phase A, acetonitrile as phase B, use 20*250mm, C18 reverse phase column, flow rate 10ml/min, carry out gradient elution, collect the target peak, analyze and detect each component, Combine the qualified components, repeat the purification of the unqualified components with other types of chromatographic columns or replace other elution systems for purification, collect and combine the components that meet the purity requirements.
  • the collected components are evaporated to remove the organic solvent, and then settled with a dilute acetic acid aqueous solution, centrifuged, the precipitate is collected, washed with water, reconstituted, filtered, lyophilized, weighed, and the yield is calculated to be ⁇ 70%. Divide and store in the frozen after labeling. Analyze the corresponding mass spectrometry data according to the molecular weight of the target peptide, and the theoretical value and the actual measured value are within plus or minus 1.
  • Example 4 In vitro activity determination of triple agonists
  • the activity of the triple agonist prepared in Examples 1, 2, and 3 was measured by a method of measuring cell activity in vitro using a cell line in which GLP-1 receptor, GCG receptor, and GIP receptor were transformed respectively.
  • the GLP-1 receptor is expressed in human renal epithelial H293 cells, while the GCG receptor and GIP receptor are expressed in Chinese hamster ovary cells CHO K1, and the activity of each part is measured using a separately transformed cell line.
  • Cisbio's cAMP-GS Dynamic Kit kit for the determination of triple agonist in vitro activity.
  • the test method used is HTRF (Homogeneous Time-Resolved Fluorescence), which requires an enzyme label equipped with an HTRF module. Instrument to complete.
  • the triple agonist prepared in Example 1 was serially diluted from 3000 nM to 1 nM; as a positive control, natural GLP-1 was also Perform 3-fold serial dilutions from 3000 nM to 1 nM.
  • the adherent cultured human renal epithelial cells H293 expressing GLP-1R were digested, centrifuged, collected, diluted to a concentration of 600,000 cells/ml, and added to the detection wells of a 384-well plate. Add 5 to each hole.
  • the triple agonists prepared in Example 1 were serially diluted from 3000 nM to 1 nM; as a positive control, natural GCG was from 1000 nM to 0.5 nM Perform a 3-fold serial dilution.
  • the adherent cultured Chinese hamster ovary cells CHO K1 expressing GCGR were digested, centrifuged, and collected, and diluted to a concentration of 600,000 cells/ml and added to the detection wells of a 384-well plate. Add 5 to the hole.
  • the triple agonists prepared in Example 1 were serially diluted from 1000 nM to 0.1 nM; as a positive control, the natural GIP was from 30 nM to 0.25 nM performs a 3-fold serial dilution.
  • the adherent cultured GIPR-expressing Chinese hamster ovary cells CHO K1 were digested, centrifuged, collected, diluted to a concentration of 600,000 cells/ml, and added to the detection wells of a 384-well plate. Add 5 to the hole.
  • the results of the assay show that the polypeptides and polypeptide-modified compounds obtained in Examples 1, 2 and 3 are not necessarily more active than a single GLP-1, GCG or GIP receptor agonist, but for GLP-1 receptor, Both GCG receptor and GIP receptor have activating activity, which is in line with the characteristics of the triple agonist described in this patent.

Abstract

提供了针对胰高血糖素样肽-1受体(GLP1-R)、胰高血糖素受体(GCGR)以及抑胃肽受体(GIPR)的三重激动剂，该三重激动剂是在天然毒蜥外泌肽(Exendin-4)氨基酸序列的基础上，至少有一个位点存在氨基酸的替换、突变或者化学修饰。本发明的三重激动剂可用于预防或治疗代谢综合征。

Description

针对胰高血糖素样肽-1受体、胰高血糖素受体、以及抑胃肽受体的三重激动剂
本申请要求于2019年12月23日提交中国专利局、申请号为201911341821.0、发明名称为“针对胰高血糖素样肽-1受体、胰高血糖素受体、以及抑胃肽受体的三重激动剂”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明涉及生物领域，具体涉及对GLP1受体，GCG受体以及GIP受体都具有活性的三重激动剂，及其相关用途。
背景技术
随着生活水平的提高，肥胖以及糖尿病已经成为非常普遍的代谢性疾病。肥胖是糖尿病的主要诱因之一 [1]，从肥胖到糖尿病的进展一般为：肥胖→糖耐量低减→2型糖尿病→难以控制的高血糖→糖尿病并发症。因此对于糖尿病的治疗，降血糖是一方面，降低体重也是必须要考虑的因素。
胰高血糖素样肽-1(GLP-1)及其类似物是目前广泛使用的非胰岛素类降糖药。GLP-1是由肠道L细胞分泌的一种多肽类激素。其通过葡萄糖依赖方式作用于胰岛β细胞，促进胰岛素的生物合成和分泌，胰岛素的升高带来明显的降糖效果；同时刺激β细胞的增殖和分化，抑制β细胞凋亡，增加胰岛β细胞数量，抑制胰高血糖素的分泌，抑制食欲及摄食，延缓胃内容物排空 [2]。
胰高血糖素(GCG)是由胰岛α细胞分泌的一种激素。与胰岛素相对抗，起着增加血糖的作用。胰高血糖素具有很强的促进糖原分解和糖异生作用，使血糖明显升高；同时可以激活脂肪酶，促进脂肪分解，又能加强脂肪酸氧化，使酮体生成增多 [3]。因此GCG虽然与降糖的目标相悖，但是却具有明显的降脂、降低体重的作用。
而抑胃肽，或称葡萄糖依赖性胰岛素释放肽(GIP)是由小肠粘膜的K细胞所产生的一类激素，其可以抑制胃酸分泌；抑制胃蛋白酶原分泌；刺激胰岛素释放；抑制胃的蠕动和排空；刺激小肠液的分泌；刺激胰高血糖素的分泌 [4]。
这三类激素的受体都属于GPCR受体家族，具有类似的蛋白结构以及结合机理，使 得设计针对两个或者三个受体的多重激动剂成为可能。针对GLP1-R、GCGR以及GIPR的三重激动剂，能够同时发挥三类激素的功能 [5，6]。GLP-1能够降低血糖，抑制食欲；GCG能够分解脂肪，降低体重，但是会升高血糖；GIP主要刺激胰岛素分泌，但是也具有刺激GCG分泌的活性。三者相互配合，依血糖浓度形成一种反馈机制，既能控制血糖，又能分解脂肪，降低体重。对于糖尿病、肥胖的治疗，三重激动剂比单纯的GLP-1类似物具有显著的优势。
发明内容
本发明的目的是提供针对胰高血糖素样肽-1受体(GLP1-R)，胰高血糖素受体(GCGR)，以及抑胃肽受体(GIPR)的三重激动剂。
本发明的另一个目的是提供编码所述的三重激动剂的基因，包括基因的重组表达载体，以及包括基因或重组表达载体的转化体。
本发明还有一个目的是提供用于合成和分离纯化制备所述的三重激动剂的方法。
本发明还有一个目的是提供用于所述的三重激动剂中某些氨基酸侧链化学修饰的方法。
本发明还有一个目的是提供用于制备含有化学修饰的所述的三重激动剂的分离纯化方法。
本发明还有一个目的是提供含有所述的三重激动剂的组合物。
本发明还有一个目的是提供用于治疗目的疾病的方法，该方法包括向受治疗对象提供所述的三重激动剂、或含有所述的三重激动剂的组合物。
本发明还有一个目的是提供用于药物制备的所述的三重激动剂或含有所述的三重激动剂的组合物的用途，即用于糖尿病、肥胖症、高血脂、高胆固醇、非酒精性脂肪肝损伤、以及由于高胆固醇血症、高血脂引起的动脉硬化、动脉粥样硬化以及冠心病等。
为实现上述目的，本发明的技术方案：
本发明提供针对胰高血糖素样肽-1受体(GLP1-R)，胰高血糖素受体(GCGR)，以及抑胃肽受体(GIPR)的具有激动活性的分离的多肽，即针对针对胰高血糖素样肽-1受体、胰高血糖素受体、以及抑胃肽受体的三重激动剂。
在一个具体实施方案中，本发明所述的三重激动剂在天然毒蜥外泌肽(Exendin-4)氨基酸序列基础上，至少有一个位点存在氨基酸的替换，突变或者化学修饰。
在另一个具体实施方案中，本发明所述的三重激动剂为包含下述通式1的氨基酸序列：
His-Xaa1-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Xaa2-Xaa3-Xaa4-Glu-Xaa5-Xaa6-Ala-Xaa7-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Xaa8-Xaa9-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Xaa10
在通式1中：
Xaa1为甘氨酸(Gly，G)或氨基异丁酸(Aib)；
Xaa2为丙氨酸(Ala，A)或精氨酸(Arg，R)或亮氨酸(Leu，L)；
Xaa3为丙氨酸(Ala，A)或谷氨酸(Glu，E)或酪氨酸(Tyr，Y)；
Xaa4为甲硫氨酸(Met，M)或亮氨酸(Leu，L)或谷氨酰胺(Gln，Q)；
Xaa5为赖氨酸(Lys，K)或带有侧链亲脂基团化学修饰的赖氨酸K-R1或K-R2；
Xaa6为谷氨酸(Glu，E)或者异亮氨酸(Ile，I)；
Xaa7为丙氨酸(Ala，A)或者缬氨酸(Val，V)；
Xaa8为异亮氨酸(Ile，I)或者精氨酸(Arg，R)；
Xaa9为天冬氨酸(Asp，D)或谷氨酸(Glu，E)；
Xaa10为丝氨酸(Ser，S)或者酰胺化的丝氨酸(S-NH 2)。
在另一个具体实施方案中，通式1中Xaa5是侧链经过化学修饰的氨基酸K-R1，其中K-R1化学结构如下：
Figure PCTCN2021075738-appb-000001
在另一个具体中实施方案，通式1中Xaa5是侧链经过化学修饰的氨基酸K-R2，其中K-R2化学结构如下：
Figure PCTCN2021075738-appb-000002
在另一个具体中实施方案，本发明所述三重激动剂选自包含表1中序号1-14任一的氨基酸序列：
表1
Figure PCTCN2021075738-appb-000003
其中，K-R1和K-R2如前所述。
在另一个具体实施方案中，本发明所述的三重激动剂至少具有以下三种活性中的一种：
I)GLP1受体的活化作用；
II)胰高血糖素受体的活化作用；以及
III)抑胃肽受体的活化作用。
在另一个具体实施方案中，与天然GLP-1，胰高血糖素或抑胃肽中任一种相比，本发明所述的三重激动剂在K残基上进行R1或R2修饰后具有更长的体内半衰期。
在另一个具体实施方案中，本发明所述的三重激动剂的C末端被酰胺化。
本发明还有一个方面是提供编码所述的三重激动剂的基因，包括该基因的重组表达载体，以及包括基因或重组表达载体的转化体。
本发明还有一个方面是提供用于合成和分离所述的三重激动剂的方法。
本发明还有一个方面是提供用于所述的三重激动剂中某些氨基酸侧链化学修饰的方法。
本发明还有一个方面是提供用于制备含有化学修饰的所述的三重激动剂的方法。
本发明还有一个方面是提供含有所述的三重激动剂的组合物，所述组合物还包括药学上可接受的载体。
在一个具体实施方案中，组合物为药学组合物。
本发明还提供所述的三重激动剂或含有所述的三重激动剂的组合物的医药用途。
在另一个具体实施方案中，本发明还提供所述的三重激动剂或含有所述的三重激动剂的组合物在制备预防和/或治疗代谢综合征疾病的药物中的用途。
在另一个具体实施方案中，本发明所述的代谢综合征包括但不限于糖尿病、肥胖症、高血压、血脂障碍、高胆固醇血症、非酒精性脂肪性肝炎、以及由于高胆固醇血症、高血脂引起的动脉硬化、动脉粥样硬化以及冠心病等等。
本发明还有一个方面是提供用于治疗目的疾病的方法，该方法包括向受治疗对象提供所述三重激动剂或含有所述三重激动剂的组合物。
发明的有益效果：
本发明的三重激动剂具有针对胰高血糖素样肽-1受体(GLP1-R)，胰高血糖素受体(GCGR)，以及抑胃肽受体(GIPR)的活性，这些活性已被证实是上述代谢综合征的治疗靶点，因此本发明所述的三重激动剂可应用于上述代谢综合征的预防或治疗药物的开发和临床治疗。
附图说明
图1：序号5多肽样品制备色谱图；
图2：序号5多肽样品UPLC分析色谱图；
图3：序号5多肽样品质谱分析图；
图4：序号6多肽样品制备色谱图；
图5：序号6多肽样品UPLC分析色谱图；
图6：序号6多肽样品质谱分析图；
图7：序号11多肽样品制备色谱图；
图8：序号11多肽样品UPLC分析色谱图；
图9：序号11多肽样品质谱分析图；
图10：序号14多肽样品制备色谱图；
图11：序号14多肽样品UPLC分析色谱图。
图12：序号14多肽样品质谱分析图。
具体实施方式
在下文中，将更详细地描述本发明。
除非本文另有定义，否则本申请中所使用的科学和技术术语应具有本领域普通技术人员通常理解的含义。通常，本文所述的与化学、分子生物学、细胞生物学、微生物学、药理学以及蛋白质和核酸化学关联使用的术语和方法是本领域中公知和常用的。
本文公开的各种要素的所有组合均属于本发明的范围。此外，本发明的范围不应受到下文提供的具体公开内容的限制。
另外，本发明提及的氨基酸根据IUPAC-IUB的命名规则缩写如下：
丙氨酸(Ala，A)，精氨酸(Arg，R)，天冬酰胺(Asn，N)，天冬氨酸(Asp，D)，半胱氨酸(Cys，C)，谷氨酸(Glu，E)谷氨酰胺(Gln，Q)，甘氨酸(Gly，G)，组氨酸(His，H)，异亮氨酸(Ile，I)，亮氨酸(Leu，L)，赖氨酸(Lys，K)，甲硫氨酸(Met，M)，苯丙氨酸(Phe，F)，脯氨酸(Pro，P)，丝氨酸(Ser，S)，苏氨酸(Thr，T)，色氨酸(Trp，W)，酪氨酸(Tyr，Y)，缬氨酸(Val，V)。
另外，本发明的多肽中的所有氨基酸残基优选为L构型。
另外，在所述序列的C端上的“-NH 2”部分表示C端上的酰胺基(-CONH 2)。
另外，本发明序列中除了天然氨基酸以外，还使用了非天然氨基酸氨基异丁酸(Aib)。
与天然配体(天然胰高血糖素、天然GLP-1和天然GIP)相比，对胰高血糖素、GLP-1和GIP受体具有显著水平的活性的三重激动剂对于胰高血糖素、GLP-1和GIP受体中的一种或多种受体、具体地两种或更多种受体、和更具体地所有三种受体，可呈现0.1％或更高、1％或更高、2％或更高、3％或更高、4％或更高、5％或更高、6％或更高、7％或更高、8％或更高、9％或更高、10％或更高、20％或更高、30％或更高、40％或更高、50％或更高、 60％或更高、70％或更高、80％或更高、90％或更高、以及100％或更高的体外活性，但不特别地限制于此。
用于测量三重激动剂的体外活性的方法可参照本发明的实施例4，但不特别限制于此。
另外，三重激动剂可以是相对于天然GLP-1、天然胰高血糖素和天然GIP中的任一个在K残基上进行R1或R2修饰后具有增加的体内半衰期的激动剂，但不特别限制于此。
另外，本文所述的多肽是选自在天然毒蜥外泌肽(Exendin-4)序列的至少一个氨基酸上的取代、修饰和其组合的变异的天然毒蜥外泌肽的类似物，但不特别限制于此。其中氨基酸的取代可包括以氨基酸取代和以非天然化合物取代两者。
更具体地，毒蜥外泌肽类似物可以是其中选自在天然毒蜥外泌肽氨基酸序列中的位置2、3、12、13、14、16、17、19、27、28和39处的氨基酸的1个或更多、2个或更多、3个或更多、4个或更多、5个或更多、6个或更多、7个或更多、8个或更多、9个或更多、10个或更多、或11个氨基酸被其他氨基酸取代的那些，但不特别限制于此。
在上述天然毒蜥外泌肽中替换的氨基酸可选自丙氨酸、精氨酸、天冬酰胺、天冬氨酸、半胱氨酸、谷氨酸、谷氨酰胺、甘氨酸、组氨酸、异亮氨酸、亮氨酸、赖氨酸、甲硫氨酸、苯丙氨酸、脯氨酸、丝氨酸、苏氨酸、色氨酸、酪氨酸、缬氨酸以及氨基异丁酸，但不特别限制于此。
另外，在上述天然毒蜥外泌肽中替换的氨基酸还可选择侧链经过亲脂性基团化学修饰的氨基酸，具体是赖氨酸(Lys，K)残基的侧链与亲脂性取代基缀合。两种亲脂性化学修饰后的赖氨酸K-R1与K-R2的化学结构如下(以下实施例中K-R1与K-R2的结构式同此处)：
Figure PCTCN2021075738-appb-000004
Figure PCTCN2021075738-appb-000005
另外，根据本发明的多肽可包括多肽其本身、其盐(例如，其药学上可接受的盐)、或其溶剂化物的所有形式。另外，多肽可以是任何药学上可接受的形式。其中，盐的种类不受特别限制，但是优选为对对象(例如，哺乳动物)安全和有效的盐，但不特别限制于此。术语“药学上可接受的”是指在不引起过度的毒性、剌激、过敏反应等的情况下，可在医药学(pharmaco-medica1)决定的范围内有效地用于预期用途的物质。术语“溶剂化物”是指根据本发明的多肽或其盐与溶剂分子之间形成的复合物。
另外，三重激动剂可以是包括选自表1中序号114的任一氨基酸序列的激动剂，以及(基本上)由选自表1中序号1-14任一的氨基酸序列组成的激动剂，但不限制于此。
另外，尽管在本发明中描述为“由特定氨基酸序列组成的多肽”但只要该肽具有与由相应的氨基酸序列组成的肽的活性相同或对应的活性，它就不排除可通过相应的氨基酸序列的上游或下游的无意义序列的添加发生的突变，或者序列内自然发生的突变，或者沉默突变来得到，并且即使当序列添加或突变存在时，它们也依然属于本发明的范围。
本文所述的三重激动剂可显著地激活胰高血糖素受体、GLP-1受体和GIP受体中的至少一种，但不特别限制于此。具体地，三重激动剂可以显著激活GLP-1受体，或额外地显著激活胰高血糖素受体和/或GIP受体，但不特别限制于此。
上述内容可适用于本发明的其他具体实施方式或方面，但不限制于此。
本发明另一方面提供编码所述的三重激动剂的多核苷酸、包含多核苷酸的重组表达载体、和包含多核苷酸或重组表达载体的转化体。
本发明的重组载体没有特别限制，只要载体可在宿主细胞中复制，可使用本领域己知的任何载体构建。用于本发明的宿主没有特别限制，只要它可表达本发明的多核昔酸。适合的宿主的实例包括诸如大肠杆菌、毕赤酵母、昆虫细胞、以及动物细胞，诸如CHO细胞等等。
另外，本发明所述的多肽既可以通过多肽固态自动合成仪合成，也可以通过基因工程技术生产。
具体地，本发明的所述的三重激动剂可通过标准合成方法、重组表达系统、或本领域己知的任何其他方法制备。因此，本发明的所述的三重激动剂可通过许多方法合成，包括例如下面描述的方法：
I)通过固相多肽自动合成仪合成目标所述的三重激动剂，然后分离和纯化最终产物的方法，或者
II)在宿主细胞中表达编码所述的三重激动剂的核酸构建体并且从宿主细胞培养物回收表达产物的方法，或者
通过I，II的任意组合获得所述的三重激动剂片段，通过连接所述的三重激动剂片段获得目标产物，并且回收目标所述的三重激动剂的方法，或者；
在更具体的实例中，目标所述的三重激动剂可通过基因工程方法生成，其包括制备编码包含融合配偶体和所述的三重激动剂的融合蛋白的融合基因，将其转化到宿主细胞中，表达融合蛋白，然后使用蛋白酶或化合物从融合蛋白中切割所述的三重激动剂然后分离。
本发明还有一个方面是提供用于所述的三重激动剂中某些氨基酸侧链化学修饰的方法。同时本发明还有一个方面是提供用于制备含有化学修饰的所述的三重激动剂的方法。
本发明涉及的氨基酸侧链化学修饰具体指的是赖氨酸侧链亲脂基团的化学修饰。本发明涉及的赖氨酸侧链化学修饰主要有两种，分别称为K-R1以及K-R2，具体化学结构差异参见如下示意图。其中R1亲脂基团侧链的化学合成方法，以及通过反应连接到赖氨酸侧链的方法参见201811653280.0。第二种情况R2亲脂基团侧链的化学合成方法，以及通过反应连接到赖氨酸侧链的方法参见CN1271086C。
本发明还有一个方面是提供含有分离所述的三重激动剂的组合物，具体来说，组合物可以是药学组合物，并且更具体地，用于预防或治疗代谢综合征的药学组合物。
如本文所用，术语“代谢综合征”是指由于慢性代谢紊乱发生的各种疾病单独或组合发生的症状。特别地，属于代谢综合征的疾病实例可包括葡萄糖耐受不良、糖尿病、肥胖症、高血压、血脂障碍、高胆固醇血症、非酒精性脂肪性肝炎、以及由于高胆固醇血症、高血脂引起的动脉硬化、动脉粥样硬化以及冠心病等，但不限制于此。
实施例1：无侧链化学修饰三重激动剂的合成制备
制备对GLP-1、GIP和GCG受体全部都显示活性的三重激动剂，其中无侧链化学修饰的部分，它们的氨基酸序列显示在下表2中。
表2
序号 氨基酸序列
1 HXQGT FTSDL SRAME KIAVR LFIEW LREGG PSSGA PPPS
2 HXQGT FTSDL SRAME KEAVR LFIEW LREGG PSSGA PPPS
3 HXQGT FTSDL SRAME KEAVR LFIEW LIEGG PSSGA PPPS
4 HXQGT FTSDL SRAME KEAVR LFIEW LIDGG PSSGA PPPS
5 HXQGT FTSDL SAAME KIAVR LFIEW LREGG PSSGA PPPS
6 HXQGT FTSDL SRAME KIAVR LFIEW LIEGG PSSGA PPPS
7 HXQGT FTSDL SRALE KIAAR LFIEW LIEGG PSSGA PPPS
8 HXQGT FTSDL SLAQE KEAVR LFIEW LREGG PSSGA PPPS
9 HXQGT FTSDL SLEQE KIAVR LFIEW LREGG PSSGA PPPS
10 HXQGT FTSDL SRYLE KEAVR LFIEW LIDGG PSSGA PPPS
注:表2所述序列中，X表示非天然氨基酸氨基异丁酸(Aib)。
无侧链化学修饰多肽的合成：以带不同氨基酸的Fmoc-AA-Wang resin为起始原料，按照合成序列进行脱保护偶联。偶联物料摩尔配比为：Fmoc-AA-Wang resin/Fmoc-AA-OH/HOBT/DIC＝1/5/6/6，活化溶剂为DMF；偶联时间为1.5h；脱保护为20％pip/DMF，时间为：5+15min，室温下反应以及脱保护。按照VS-P、VS-C系列多肽序列合成直至结束，干燥得到肽树脂。
配制比例为TFA/PhSMe/PhOMe/EDT＝90/5/2/3的裂解液裂解，使用量为1g肽树脂使用10ml裂解液裂解。低温下加入肽树脂先反应10min，后转移至室温下反应2h；过滤，滤液加入冰甲基叔丁基醚沉降，按照1ml裂解液使用10ml冰甲基叔丁基醚沉降1次，再使用相同量的冰甲基叔丁基醚洗涤5次，离心，得到多肽粗品。
合成样品的处理：合成的粗肽，真空干燥完全，用磷酸三乙胺缓冲液(pH＝1.8-3.0)水溶液溶解，配置成约5mg/ml样品溶液，0.45微米滤膜过滤，滤液用稀氨水溶液调pH至样品等电点，离心，收集沉淀。沉淀再用磷酸三乙胺缓冲液复溶，0.45微米滤膜过滤，备用。
多肽的纯化：用90％的20mM磷酸三乙胺缓冲液(pH＝1.80)+10％乙腈作为A相，90％乙腈+10％20mM磷酸三乙胺缓冲液为B相，用20*250mm，C18反相柱，流速10ml/min，进行梯度洗脱，收集目的峰，分析检测各组分，合并合格组分，不合格组分重复纯化或更换其它洗脱体系进行纯化，收集并合并符合纯度要求的组分。将收集组分通过旋 蒸去掉乙腈，然后用稀氨水调沉，离心，收集沉淀，水洗后用稀醋酸水溶液溶解，过滤，将滤液冻干，称重后计算收率≥40％。分装冷冻保存。根据目标多肽分子量分析相应的质谱数据，理论值与实测值范围为正负1以内。
部分多肽制备反向色谱图，冻干后纯品UPLC分析色谱图以及质谱分析图如图1-6所示。
实施例2：带R1侧链化学修饰的三重激动剂的合成制备
带有R1侧链化学修饰的三重激动剂氨基酸序列见下表：
表3
Figure PCTCN2021075738-appb-000006
注:表3所述序列中，X表示非天然氨基酸氨基异丁酸(Aib)；-NH 2表示酰胺化的多肽C端(-CONH 2)；K-R1以及K-R2分别表示赖氨酸侧链的两种亲脂基团化学修饰，具体化学结构见上文所述。
带R1侧链多肽的合成：以Rink Amide-MBHA resin为起始原料，按照合成序列进行脱保护偶联。偶联物料摩尔配比为：Rink Amide-MBHA resin/Fmoc-AA-OH/HOBT/DIC＝1/5/6/6，活化溶剂为DMF；偶联时间为1.5h；脱保护为20％pip/DMF，时间为：5+15min，室温下反应以及脱保护。按照VS-CA系列多肽序列合成直至结束，干燥得到肽树脂。
配制比例为TFA/PhSMe/PhOMe/EDT＝90/5/2/3的裂解液裂解，每1g肽树脂使用10ml裂解液裂解。低温下加入肽树脂10min，后转移至室温下反应2h；过滤，滤液加入冰甲基叔丁基醚沉降，按照1ml裂解液使用10ml冰甲基叔丁基醚沉降1次，再使用相同量的冰甲基叔丁基醚洗涤5次，离心，干燥得到多肽粗品。
将多肽粗品加入0.25M的pH＝11.65的磷酸三乙胺缓冲溶液中搅拌溶解(浓度为10mg多肽/ml缓冲液)；冰浴冷却至0-5℃，加入溶于N-甲基吡咯烷酮的R1侧链(100mg/ml)3.0eq，0-5℃下反应10min结束，得到粗肽。R1侧链合成方法详见专利201811653280.0。
合成样品的处理：合成的粗肽，真空干燥完全后用稀碳酸氢铵溶液溶解，配置成约5mg/ml样品溶液，0.45微米滤膜过滤，备用。
多肽的纯化：用含有1％醋酸，20％乙醇的水溶液为A相，含有1％醋酸，80％乙醇的水溶液为B相，用20*250mm，PS反相柱，流速10ml/min，进行梯度洗脱，收集目的峰，分析检测各组分，合并合格组分，不合格组分用C18反向柱重复纯化或更换其它洗脱体系进行纯化，收集并合并符合纯度要求的组分。将收集组分通过旋蒸去掉有机溶剂，然后用稀醋酸水溶液调沉，离心，收集沉淀，水洗，复溶，过滤，冻干，称重后计算收率≥70％。分装后冷冻保存。根据目标多肽分子量分析相应的质谱数据，理论值与实测值范围为正负1以内。
部分多肽制备反向色谱图，冻干后纯品UPLC分析色谱图以及质谱分析图如图7-9所示。
实施例3：带R2侧链化学修饰的三重激动剂的合成制备
表4
Figure PCTCN2021075738-appb-000007
注:表4所述序列中，X表示非天然氨基酸氨基异丁酸(Aib)；-NH 2表示酰胺化的多肽C端(-CONH 2)；K-R1以及K-R2分别表示赖氨酸侧链的两种亲脂基团化学修饰，具体化学结构见上文所述。
带R2侧链多肽的合成：以Rink Amide-MBHA resin为起始原料，按照合成序列进行脱保护偶联。偶联物料摩尔配比为：Rink Amide-MBHA resin/Fmoc-AA-OH/HOBT/DIC＝1/5/6/6，活化溶剂为DMF；偶联时间为1.5h；脱保护为20％pip/DMF，时间为：5+15min，室温下反应以及脱保护。按照VS-CA系列多肽序列合成直至结束，干燥得到肽树脂。
配制比例为TFA/PhSMe/PhOMe/EDT＝90/5/2/3的裂解液裂解，每1g肽树脂使用10ml 裂解液裂解。低温下加入肽树脂10min，后转移至室温下反应2h；过滤，滤液加入冰甲基叔丁基醚沉降，按照1ml裂解液使用10ml冰甲基叔丁基醚沉降1次，再使用相同量的冰甲基叔丁基醚洗涤5次，离心，干燥得到多肽粗品。
将多肽粗品加入0.25M的pH＝11.65的磷酸三乙胺缓冲溶液中搅拌溶解(浓度为10mg多肽/ml缓冲液)；冰浴冷却至0-5℃，加入溶于N-甲基吡咯烷酮的R2侧链(100mg/ml)1.5eq，0-5℃下反应10min结束，得到粗肽。R2侧链合成方法详见专利CN1271086C。
合成样品的处理：合成的粗肽，真空干燥后用纯化水配置成约5mg/ml样品溶液，用稀氨水调pH至7.0-9.0之间，0.45微米滤膜过滤，备用。
多肽的纯化：用含有80mM醋酸铵的水溶液为A相，乙腈为B相，用20*250mm，C18反相柱，流速10ml/min，进行梯度洗脱，收集目的峰，分析检测各组分，合并合格组分，不合格组分用其他类型色谱柱重复纯化或更换其它洗脱体系进行纯化，收集并合并符合纯度要求的组分。将收集组分通过旋蒸去掉有机溶剂，然后用稀醋酸水溶液调沉，离心，收集沉淀，水洗，复溶，过滤，冻干，称重后计算收率≥70％。分装，贴标签后冷冻保存。根据目标多肽分子量分析相应的质谱数据，理论值与实测值范围为正负1以内。
部分多肽制备反向色谱图，冻干后纯品UPLC分析色谱图以及质谱分析图如图10-12所示。
实施例4：三重激动剂的体外活性测定
实施例1、2、3中制备的三重激动剂的活性通过使用其中GLP-1受体、GCG受体和GIP受体被分别转化的细胞系测量体外细胞活性的方法进行测量。其中GLP-1受体被表达在人肾上皮H293细胞中，而GCG受体及GIP受体被表达在中国仓鼠卵巢细胞CHO K1中，各部分的活性使用分别转化的细胞系测量。对于三重激动剂体外活性的测定，使用Cisbio公司的cAMP-GS Dynamic Kit试剂盒完成，采用的测试方法是HTRF(均相时间分辨荧光法，Homogeneous Time-Resolved Fluorescence)，需要配备HTRF模块的酶标仪来完成。
为了测量实施例1、2、3中制备的三重激动剂的GLP-1活性，对实施例1中制备的三重激动剂从3000nM至1nM进行3倍连续稀释；作为阳性对照，天然GLP-1也从3000nM至1nM进行3倍连续稀释。为了测试三重激动剂的GLP-1R活性，将贴壁培养的表达GLP-1R的人肾上皮细胞H293消化、离心、收集之后，稀释到600000个/ml的浓度分别加入到384孔板的检测孔中，每孔加入5 1。随后加入稀释后的三重激动剂，每孔5 1，混 合后37℃培养90min。按照CAMP-GS Dynamic Kit试剂盒的要求，向384孔板检测孔中分别先后加入稀释后的cAMP-d2试剂以及cAMP-Cryptate试剂，混合均匀后室温避光放置60min。最后将384孔板置于配备HTRF模块的酶标仪中度数，通过累积的cAMP计算EC50值，并且将这些值相互比较。与人GLP-1相比的相对效价显示在以下表2中。
为了测量实施例1、2、3中制备的三重激动剂的GCG活性，对实施例1中制备的三重激动剂从3000nM至1nM进行3倍连续稀释；作为阳性对照，天然GCG从1000nM至0.5nM进行3倍连续稀释。为了测试三重激动剂的GCG活性，将贴壁培养的表达GCGR的中国仓鼠卵巢细胞CHO K1消化、离心、收集之后，稀释到600000个/ml的浓度分别加入到384孔板的检测孔中，每孔加入5 1。随后加入稀释后的三重激动剂，每孔5 1，混合后37℃培养90min。按照CAMP-GS Dynamic Kit试剂盒的要求，向384孔板检测孔中分别先后加入稀释后的cAMP-d2试剂以及cAMP-Cryptate试剂，混合均匀后室温避光放置60min。最后将384孔板置于配备HTRF模块的酶标仪中度数，通过累积的cAMP计算EC50值，并且将这些值相互比较。与人GCG相比的相对效价显示在以下表2中。
为了测量实施例1、2、3中制备的三重激动剂的GIP活性，对实施例1中制备的三重激动剂从1000nM至0.1nM进行3倍连续稀释；作为阳性对照，天然GIP从30nM至0.25nM进行3倍连续稀释。为了测试三重激动剂的GIPR活性，将贴壁培养的表达GIPR的中国仓鼠卵巢细胞CHO K1消化、离心、收集之后，稀释到600000个/ml的浓度分别加入到384孔板的检测孔中，每孔加入5 1。随后加入稀释后的三重激动剂，每孔5 1，混合后37℃培养90min。按照CAMP-GS Dynamic Kit试剂盒的要求，向384孔板检测孔中分别先后加入稀释后的cAMP-d2试剂以及cAMP-Cryptate试剂，混合均匀后室温避光放置60min。最后将384孔板置于配备HTRF模块的酶标仪中度数，通过累积的cAMP计算EC50值，并且将这些值相互比较。与人GIP相比的相对效价显示在以下表5中。测定的结果显示实施例1,2和3中得到的多肽及多肽修饰化合物尽管相对于单一的GLP-1、GCG或GIP受体激动剂的活性不一定更强，但对于GLP-1受体、GCG受体和GIP受体三者均有激活活性，符合本专利所述三重激动剂的特点，由于这三个受体已被证实是糖尿病、肥胖症、高血压、血脂障碍、高胆固醇血症、非酒精性脂肪性肝炎、由于高胆固醇血症、高血脂引起的动脉硬化、动脉粥样硬化以及冠心病等代谢综合征的治疗靶点，现有已上市药物均为这三个靶点的单一激动剂，因此本发明所述的三重激动剂可应用于这些代谢综合征治疗药物的开发和临床治疗，体外研究数据初步支持了这一效果，将为这类疾病的新一代治疗药物的开发 提供依据。
表5
Figure PCTCN2021075738-appb-000008
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Claims (8)

  1. 针对胰高血糖素样肽-1受体、胰高血糖素受体、以及抑胃肽受体的三重激动剂，其特征在于，其氨基酸序列符合以下通式1：
    His-Xaa1-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Xaa2-Xaa3-Xaa4-Glu-Xaa5-Xaa6-Ala-Xaa7-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Xaa8-Xaa9-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Xaa10
    其中，在通式1中：
    Xaa1为甘氨酸(Gly，G)或氨基异丁酸(Aib)；
    Xaa2为丙氨酸(Ala，A)或精氨酸(Arg，R)或亮氨酸(Leu，L)；
    Xaa3为丙氨酸(Ala，A)或谷氨酸(Glu，E)或酪氨酸(Tyr，Y)；
    Xaa4为甲硫氨酸(Met，M)或亮氨酸(Leu，L)或谷氨酰胺(Gln，Q)；
    Xaa5为赖氨酸(Lys，K)或带有侧链亲脂基团化学修饰的赖氨酸K-R1或K-R2；
    Xaa6为谷氨酸(Glu，E)或者异亮氨酸(Ile，I)；
    Xaa7为丙氨酸(Ala，A)或者缬氨酸(Val，V)；
    Xaa8为异亮氨酸(Ile，I)或者精氨酸(Arg，R)；
    Xaa9为天冬氨酸(Asp，D)或谷氨酸(Glu，E)；
    Xaa10为丝氨酸(Ser，S)或者酰胺化的丝氨酸(S-NH 2)。
  2. 根据权利要求1所述的三重激动剂，其特征在于，Xaa5中，K-R1的化学结构如下：
    Figure PCTCN2021075738-appb-100001
    K-R2的化学结构如下：
    Figure PCTCN2021075738-appb-100002
  3. 根据权利要求1所述的三重激动剂，其特征在于，其氨基酸序列选自下表中序号1-14中的任一种：
    Figure PCTCN2021075738-appb-100003
    其中，K-R1和K-R2如权利要求2所述。
  4. 编码权利要求1-3的任何一项所述的三重激动剂的基因。
  5. 包含权利要求1-3的任何一项所述的三重激动剂的重组表达载体或转化体。
  6. 包含权利要求1-3的任何一项所述的三重激动剂的组合物，所述组合物还包含药学上可接受的载体。
  7. 权利要求1-3的任何一项所述的三重激动剂、权利要求4所述的基因、权利要求5所述的重组表达载体或转化体、权利要求6所述的组合物在治疗和/或预防代谢综合征中的应用。
  8. 根据权利要求7所述的应用，其特征在于，其中所述代谢综合征包括糖尿病，肥胖症，高血压，血脂障碍，高胆固醇血症，非酒精性脂肪性肝炎，由于高胆固醇血症、高血脂引起的动脉硬化和冠心病。
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