CN111234000B - Ai Saina peptide analogues - Google Patents
Ai Saina peptide analogues Download PDFInfo
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- CN111234000B CN111234000B CN201811432450.2A CN201811432450A CN111234000B CN 111234000 B CN111234000 B CN 111234000B CN 201811432450 A CN201811432450 A CN 201811432450A CN 111234000 B CN111234000 B CN 111234000B
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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
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/22—Hormones
- A61K38/26—Glucagons
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- 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
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- 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
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P5/00—Drugs for disorders of the endocrine system
- A61P5/48—Drugs for disorders of the endocrine system of the pancreatic hormones
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- C—CHEMISTRY; METALLURGY
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- 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
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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
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- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
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- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
Abstract
The invention belongs to the field of biological medicine, and particularly discloses Ai Saina peptide analogues. The invention designs a plurality of amino acid structures at the C end of the amino acid sequence of the Ai Saina peptide Exendin-4, comprising the addition or deletion of amino acid, the mutation of the amino acid sequence and the addition of sites available for site-directed modification to obtain Ai Saina peptide analogues. The activity of the analogue for stimulating the islet tumor cells of mice to secrete insulin is obviously higher than that of Ai Saina peptide Ex-4, and the in-vivo hypoglycemic activity of a diabetes model mouse C57BL/KsJ-db/db is also obviously better than that of Ai Saina peptide Ex-4, so that the analogue can be used for preventing, treating or relieving diabetes, obesity and/or related complications thereof and other diseases.
Description
Technical Field
The invention belongs to the field of biological medicine, and particularly relates to Ai Saina peptide analogues.
Background
Mammalian GLP-1 is derived from a protoglucagon-like peptide secreted by L cells of the intestinal mucosa, whereas natural analogs of GLP-1, exenatide, developed by Amylin, inc., are found in saliva secretions of exendins, with amino acid sequences that share sequence similarity to several members of the GLP family, have 53% homology to human GLP-1 (7-36).
The expression and activity of human GLP-1 (7-36) in vivo is strictly regulated, and when the second Ala at the N end of the human GLP-1 is hydrolyzed by Dipeptidylpeptidase (DPP), inactive GLP-1 (9-36) is formed, and the metabolite is also an in vivo natural antagonist of GLP-1R. Thus, natural human active GLP-1 has a very short half-life in vivo and a metabolism rate of 2min; in addition, in physiological state, GLP-1 is mainly excreted through kidney, so that clinical application of human GLP-1 is limited. The second amino acid Gly of the non-human artificially synthesized Exenatide is different from Ala of human GLP-1, so that degradation of dipeptidyl peptidase can be effectively resisted; the rigid (PSSGAPPPS) amino acid sequence of the C end of Exenatide can increase the stability of the polypeptide, and the blood sugar reducing capability of Exenatide in vivo is about 1000 times stronger than GLP-1.
Ai Saina peptide (also known as Exenatide or Exenatide, or Ex-4, trade name Byetta) is a polypeptide composed of 39 amino acids and having a molecular weight of 4186.6 and a molecular formula of C 184 H 282 N 50 O 60 S, CAS number 141758-74-9, amino acid sequence is shown below.
His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH 2 The method comprises the steps of carrying out a first treatment on the surface of the Produced and sold by Amylin pharmaceuticals and Eli Lilly. Ai Saina peptide is approved by FDA in 4 2005 and belongs to medicinal subcutaneous injection preparation, which has the functions of promoting glucose-dependent insulin secretion, recovering first-phase insulin secretion, inhibiting glucagon secretion, slowing down the emptying of gastric contents, improving the functions of pancreatic beta cells and the like, and is very suitable for treating type II diabetes; for example, for improving and controlling the glycemia in patients with type II diabetes who are not ideal for metformin and sulfonylurea drug therapy.
Exenatide peptide secondary structure: the Exenatide has irregular curls at the N end, the middle parts are alternately arranged with amino acid residues with side chains with opposite charges on the same side surface, a helix is formed through a salt bridge or a polar hydrogen bond, and the C end is hydrophilic Trp-Cage. The interaction mechanism of Exenatide and GLP-1 receptor has been studied clearly.
A series of different structural designs have been developed to extend the structural half-life of GLP-1 analogues and increase the bioactivity. CN1384755 discloses a novel Exendin agonist preparation and a method for administration thereof, and discloses a compound structure of Exenatide and a preparation method thereof. CN102532303 discloses the modification of the amino group of lysine or the amino group of the N-terminal histidine residue in Exenatide with methoxypolyethylene glycol. CN200980111088 discloses fatty acid-PEG-Exenatide. Patent CN101125207 reports the structural modification of Exenatide to a short peptide. CN105753963a discloses Exenatide analogs with single or multiple point amino acid mutations. CN102397558 discloses that some amino acids in Exenatide are substituted with cysteines and modified with PEG or PEG with methyl substituted terminal.
Despite the efforts made in the development of long-acting pharmaceutical products of Ai Saina peptide analogs, the Ai Saina peptide analogs currently on the market have poor stability and low efficacy, and as the structural and active basis for the development of long-acting pharmaceutical products, innovative modification and research of Ai Saina peptide analogs remains a very important topic.
Based on the above analytical study, we selected Ai Saina peptide as the modified polypeptide sequence of the invention, combined with computer model calculation and combination test, focused on modification of the amino acid sequence at the C-terminal thereof, while adding modified active site for long-acting drug development.
Disclosure of Invention
The invention aims to overcome the defects of the prior art, provide Ai Saina peptide analogues which have long half-life, good stability and high activity and can simultaneously maintain good hypoglycemic effect and low toxicity, and simultaneously provide the application of Ai Saina peptide analogues in preparing medicaments for treating diabetes, obesity and/or complications.
The invention comprises the following specific contents:
the invention provides Ai Saina peptide analogues, which are characterized in that amino acids from 37 th to 45 th positions of an amino acid sequence of Ai Saina peptide Ex-4 are replaced to obtain Ai Saina peptide analogues; the amino acid sequence is shown as SEQ ID NO. 1, comprising:
His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Xaa37-Xaa38-Xaa39-Xaa40-Xaa41-Xaa42-Xaa43-Xaa44-Xaa45，
wherein Xaa37 is Pro or Gly;
xaa38 is Ser or Gly;
xaa39 is Ser or Gly;
xaa40 is Gly or Ala;
xaa41 is Ala or Thr;
xaa42 is Gly;
xaa43 is Gly;
xaa44 is Ser;
xaa45 is Cys.
Further preferably, the amino acid sequence of the preferred Exenatide analogues is shown in SEQ.ID NO: 2-7:
wherein Xaa37 is Gly, xaa38 is Ser, xaa39 is Gly, xaa40 is Gly, xaa41 is Thr, xaa42 is Gly, xaa43 is Gly, xaa44 is Ser, xaa45 is Cys; the Ai Saina peptide analogue Ex-4i is obtained, and the amino acid sequence of the analogue is shown as SEQ ID NO. 2.
His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Gly-Ser-Gly-Gly-Thr-Gly-Gly-Ser-Cys(SEQ.IDNO:2)。
Wherein Xaa37 is Pro, xaa38 is Gly, xaa39 is Gly, xaa40 is Gly, xaa41 is Thr, xaa42 is Gly, xaa43 is Gly, xaa44 is Ser, xaa45 is Cys; the Ai Saina peptide analogue Ex-4ii is obtained, and the amino acid sequence of the Ai Saina peptide analogue is shown as SEQ ID NO. 3.
His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Gly-Gly-Gly-Thr-Gly-Gly-Ser-Cys(SEQ.IDNO:3)。
Wherein Xaa37 is Pro, xaa38 is Ser, xaa39 is Gly, xaa40 is Gly, xaa41 is Thr Xaa42 is Gly, xaa43 is Gly, xaa44 is Ser, xaa45 is Cys; the Ai Saina peptide analogue Ex-4iii is obtained, and the amino acid sequence of the analogue is shown as SEQ ID NO. 4.
His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Gly-Gly-Thr-Gly-Gly-Ser-Cys(SEQ.IDNO:4)。
Wherein Xaa37 is Pro, xaa38 is Ser, xaa39 is Ser, xaa40 is Gly, xaa41 is Thr, xaa42 is Gly, xaa43 is Gly, xaa44 is Ser, xaa45 is Cys; the Ai Saina peptide analogue Ex-4iv is obtained, and the amino acid sequence of the Ai Saina peptide analogue is shown as SEQ ID NO. 5.
His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Thr-Gly-Gly-Ser-Cys(SEQ.ID NO:5)。
Wherein Xaa37 is Pro, xaa38 is Ser, xaa39 is Ser, xaa40 is Ala, xaa41 is Thr, xaa42 is Gly, xaa43 is Gly, xaa44 is Ser, xaa45 is Cys; the Ai Saina peptide analogue Ex-4v is obtained, and the amino acid sequence of the analogue is shown as SEQ ID NO. 6.
His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Ala-Thr-Gly-Gly-Ser-Cys(SEQ.ID NO:6)。
Wherein Xaa37 is Pro, xaa38 is Ser, xaa39 is Ser, xaa40 is Gly, xaa41 is Ala, xaa42 is Gly, xaa43 is Gly, xaa44 is Ser, xaa45 is Cys; the Ai Saina peptide analogue Ex-4vi is obtained, and the amino acid sequence of the analogue is shown as SEQ ID NO. 7.
His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Gly-Gly-Ser-Cys(SEQ.IDNO:7)。
The C-terminal amidation of the Ai Saina peptide analogs of the present invention is identical to that of the native GLP-1 and Ai Saina peptides.
It is another object of the present invention to provide the use of the Ai Saina peptide analogues of the present invention for the manufacture of a medicament for the treatment of diabetes, obesity and/or related complications.
For the above application, the medicament contains the Ai Saina peptide analogue and a pharmaceutically acceptable carrier.
The Ai Saina peptide analogs of the invention may be administered as a single drug or may be administered in combination with other drugs.
The Ai Saina peptide analogs of the invention can be salified, including a variety of inorganic or organic salts, such as hydrochloride, hydrobromide, phosphate, sulfate, maleate, oxalate, citrate, tartrate, fumarate, mandelate, and lactate; salifying with various inorganic and organic bases such as sodium hydroxide, tris-hydroxymethyl-aminomethane and N-methyl-glucamine.
The Ai Saina peptide analogs of the invention may be used alone or in pharmaceutical compositions. The pharmaceutical composition comprises the Ai Saina peptide analogue or the pharmaceutically acceptable salt thereof and the pharmaceutically acceptable carrier as active ingredients. The "pharmaceutically acceptable carrier" does not destroy the pharmaceutical activity of the compounds of the invention and their useful salts, while its effective amount is non-toxic to humans. "pharmaceutically acceptable carrier" may use, but is not limited to: ion exchange materials, aluminum stearate, lecithin, serum proteins for pharmaceutical preparations, saturated vegetable fatty acids, cellulosic materials, ethylene-polyoxyethylene-block polymers, cyclodextrins or chemically modified derivatives or other soluble derivatives, and the like.
Other pharmaceutically acceptable excipients, fillers such as lactose anhydrous, starch, lactose beads and glucose, binders such as microcrystalline cellulose, disintegrants such as croscarmellose sodium, croscarmellose starch, low substituted hydroxypropylcellulose, lubricants such as magnesium stearate, absorption promoters, excipients, solubilizers, colorants, etc. may also be added to the pharmaceutical compositions of the present invention.
The Ai Saina peptide analogues of the invention described above, or pharmaceutically acceptable salts thereof, and the pharmaceutical compositions may be administered by enteral or parenteral routes. Parenteral routes include subcutaneous, intradermal, intramuscular, nasal, mucosal administration or inhalation. The preparation can be used for the development of injection, cream, ointment, patch, aerosol, etc.
The Ai Saina peptide analogues or pharmaceutically acceptable salts thereof and pharmaceutical compositions of the invention can be used in single or combination therapy of related diseases, as would be understood by those skilled in the art.
The present invention is not limited to the methods for preparing the Ai Saina peptide Ex-4 and Ai Saina peptide analogs, and chemical solid phase synthesis methods and gene expression methods can be used. One embodiment of the present invention employs Fmoc solid phase synthesis.
The pharmaceutical test results show that the activity of 32 Ai Saina peptides similar to the activity of stimulating the mouse islet tumor cells RIN-m5F to release cAMP in vitro is obviously higher than that of the unmutated Ai Saina peptide; and the hypoglycemic effect of the subcutaneous injection on db/db model mice is obviously better than Ai Saina peptide. Thus, the Ai Saina peptide analogs can be used to better prevent, treat or alleviate diabetes, obesity and/or complications.
The difference between the 32 Ai Saina peptide analogs of the invention and the unmutated Ai Saina peptide was analyzed using MOE software (Molecular Operating Environment, chemical Computing Group, montreal, canada), and the results indicate that the interaction and bond formation number between the 32 Ai Saina peptide analogs and the receptor of the invention were changed, preferably, the C-terminal amino acid sequence of the 6 Ai Saina peptide analogs Ex-4 (i-vi) formed a novel binding effect, which stabilized the polypeptide alpha helix structure, promoted its binding to the active core structure domain, and improved the bioactivity.
The Ai Saina peptide analogs contemplated by the present invention all exhibit enhanced activity. The Ai Saina peptide Ex-4 and Ai Saina peptide analogs stimulated cAMP release activity in murine islet tumor cells RIN-m5F at different drug concentrations. In vitro biological activity increases in a dose-dependent manner. Preferably, the Ai Saina peptide analog Ex-4 (i-vi) has a higher activity to stimulate cAMP release from the RIN-5mF cells of the pancreatic islet tumors of the mice than other analogs. More preferably, the Ai Saina peptide analogs Ex-4ii, ex-4iii and Ex-4iv have higher cAMP-releasing activity in murine islet tumor cells RIN-m5F than the Ai Saina peptide analogs Ex-4i, ex-4v and Ex-4 vi.
Ex-4 and Ex-4 (i-vi) groups were subcutaneously administered for 0.5h to significantly lower blood glucose levels (P < 0.01); the hypoglycemic effect of Ex-4 can be maintained for 8 hours after single administration, and the blood sugar concentration is obviously increased after 12 hours of administration. The hypoglycemic effect of Ex-4 (i-vi) was maintained for 12h in a single administration, and the blood glucose level of Ex-4 (i-vi) was still lower than that of the model group (P < 0.05) in a single administration for 12 h. Therefore, the six analogues of Ex-4 (i-vi) have the effect of reducing blood sugar, and have longer effect of reducing blood sugar compared with the Ex-4, and particularly the effect of reducing blood sugar of Ex-4ii, ex-4iii and Ex-4iv is more remarkable. Ex-4 (i-vi) has stronger activity than Ex-4, longer efficacy maintaining time and longer half-life.
Compared with Ex-4, the Ai Saina peptide analogue provided by the invention is obtained through an in-vivo hypoglycemic effect test of a db/db mouse with a type II diabetes model, and the Ai Saina peptide and the analogue thereof can obviously reduce the blood sugar level after being subcutaneously administrated for 0.5 h; the hypoglycemic effect of Ai Saina peptide can be maintained for 8 hours after single administration, the blood sugar concentration is obviously increased after 12 hours of administration, and the Ai Saina peptide analogue has high hypoglycemic maintenance time Yu Aisai nano peptide Ex-4 and the hypoglycemic maintenance time reaches more than 10 hours. Preferably, the hypoglycemic effect of the Ai Saina peptide analogue Ex-4 (i-vi) was maintained for 12h in a single administration and the blood glucose level was still maintained lower in the Ex-4 (i-vi) group than in the model group (P < 0.05) in a single administration for 12 h. Therefore, the Ai Saina peptide analogues have the effect of reducing blood sugar, and have longer blood sugar reducing effect compared with Ai Saina peptide Ex-4. Ai Saina peptide analogs are more active than Ai Saina peptides, have longer duration of efficacy and have a longer half-life.
The Ai Saina peptide analogue designed by the invention has long half-life period, good stability and high activity, and can simultaneously maintain good hypoglycemic effect and low toxicity.
Terminology
In the present specification, the term "Ai Saina peptide" is a functional analogue of glucagon-like peptide-1 isolated from the salivary glands of exendins inhabiting the southwest united states. The Ai Saina peptide, which is academic name "Ai Saina peptide-4", as a physiologically active peptide consisting of 39 amino acids, has 53% amino acid similarity to glucagon-like peptide-1 existing in human living body. The amino acid sequence of the Ai Saina peptide, which is denoted Ex-4 in the following examples, is shown as SEQ ID NO. 1.
In the present specification, the term Ai Saina peptide analogue means a physiologically active peptide consisting of still 39 amino acids, in which the C-terminal rigid amino acid sequence of Ai Saina peptide-4 is deleted and replaced by another flexible amino acid sequence. The amino acid sequence of the preferred Ai Saina peptide analog Ex-4 (i-vi) is shown in SEQ ID NO. 2-7. +
Drawings
FIG. 1 is a schematic diagram of the interaction of Ai Saina peptide Ex-4 with G protein coupled receptors;
FIG. 2 is a graph showing the binding pattern of the C-terminal end of the Ai Saina peptide Ex-4 to the G protein-coupled receptor;
FIG. 3 is a schematic representation of the interaction of the Ai Saina peptide analog Ex4iii with G protein-coupled receptors;
FIG. 4 is a diagram showing the binding pattern of the Ai Saina peptide analog Ex4 iiiIC terminal to the G protein-coupled receptor;
FIG. 5 is the cAMP releasing activity of Ai Saina peptide Ex-4 and its analog Ex-4 (i-vi) on mouse islet tumor cells RIN-m 5F.
Detailed Description
The present invention will be described in more detail with reference to examples. These examples are only for more specifically explaining the present invention, and the scope of the present invention is not limited to these examples, as will be apparent to those of ordinary skill in the art to which the present invention pertains, in view of the gist of the present invention.
Example 1 preparation of Ai Saina peptide Ex-4 and Ai Saina peptide analogs
This example uses Fmoc solid phase synthesis to synthesize one of the Ai Saina peptide analogues of the invention, and the synthesis of the other analogues is similar.
Synthesis process
Fmoc-Rink resin was selected for synthesis using Fmoc synthesis. The synthesis steps are as follows:
19 Fmoc-amino acid raw materials with side chain protecting groups, solid phase synthesis, side chain protecting group removal, HPLC purification, freeze drying and Ai Saina peptide analogues.
Specifically, the method comprises the following steps:
1. amino acid monomers
2. Solid phase synthesis and preparation process of polypeptide crude product
The HOBt method is adopted to activate amino acid, and the amino acid is connected to amino resin according to the sequence, and 39 steps of synthesis are carried out.
(1) Instrument apparatus:
CS536XT vertical polypeptide synthesizer and magnetic stirrer.
(2) Reagent:
(3) Solid phase synthesis operation: 5.5g of resin (resin loading rate: 0.45 mmol/g) was weighed, poured into a reactor of a polypeptide synthesizer, and 10mmol of the corresponding amino acid with protecting group was weighed from C-terminal to N-terminal according to the amino acid sequence of Ai Saina peptide analogue, and placed in the synthesizer. Under the room temperature condition, the 39 steps of synthesis reaction are automatically completed according to the computer program.
After the synthesis is finished, the polypeptide resin with the side chain protecting group is obtained. The polypeptide resin was removed and placed in a vacuum dryer at a drying temperature of 30℃and dried to a constant weight (+ -0.2 g).
(4) Deprotection and precipitation
Placing the Ai Saina peptide analogue polypeptide resin with the protecting group into a triangular flask with a plug, adding a cracking reagent TFA/water/TIS/EDT=94:2:2:2 (V/V) according to the ratio of 13 ml/g of the peptide resin, and stirring and reacting for 4 hours at the constant temperature of 25 ℃; the filtrate was filtered and collected, the resin was washed with a small amount of trifluoroacetic acid, and the collected liquids were combined by filtration. 500mL of glacial diethyl ether (-10 ℃) was added dropwise with stirring to give a white precipitate, the crude product was filtered, washed with a small amount of glacial diethyl ether, and the crude product was dried overnight in a vacuum desiccator.
3. HPLC purification
(1) Instrument and reagent:
(2) Purification
The Ai Saina peptide analogue initial product trifluoroacetate solution is prepared by HPLC reversed phase purification, and the purity is more than 98%.
(1) Chromatographic column: 50mm×300mm RP-18 10 μm
(2) Mobile phase:
a:0.1% aqueous trifluoroacetic acid solution
B:0.1% acetonitrile solution of trifluoroacetic acid
(3) Loading solution: the crude polypeptide was prepared in a solution of 10.0mg/ml in 0.1% trifluoroacetic acid in water and passed through a 0.22 μm filter.
(4) Elution conditions: gradient elution was performed at a flow rate of 50ml/min, with UV 215nm detection, and the elution gradient is as follows:
(5) sample collection: and collecting effluent with main peak purity greater than 98% to obtain the eluent.
(6) Acetonitrile removal:
pouring the collected eluent into a rotary evaporation bottle, rotary evaporating at 25 ℃ and-0.099 Mpa, removing all acetonitrile, filtering the residual liquid through a 0.22 μm filter membrane, and keeping the residual liquid for freeze-drying.
4. Freeze drying
(1) Instrument apparatus: GOLD-SIM vertical freeze dryer
(2) The operation is as follows:
the Ai Saina peptide analog trifluoroacetic acid aqueous solution is poured into a sample tray of a vacuum freeze dryer, and freeze-dried according to a computer program to obtain the required compound.
Ai Saina peptide Ex-4 and other Ai Saina peptide analogs were prepared as described above.
Example 2 in silico assisted polypeptide screening
The interaction of Ai Saina peptide Ex-4 and Ai Saina peptide analogs with G protein coupled receptors was analyzed using MOE software. The results showed enhanced interaction between the 32 Ai Saina peptide analogs and the receptor binding domain compared to Ai Saina peptide Ex-4; an increased number of interactions between the Ai Saina peptide analog and the receptor; preferably the 6 Ai Saina peptide analogues Ex-4 (i-vi) form new interactions between the C-terminal end of the polypeptide and the receptor, including new hydrogen bonds, new hydrophobic interactions and new ionic bonds. The existence of the new bonds can lead the active alpha helical structure to be more stable, and the binding force of the receptor active domain to be improved, thereby improving the activity.
MOE software analysis showed that the introduced flexible amino acid sequence cross-linking region or flexible Linker replacement Ai Saina peptide analog formed a new interaction between the C-terminal and the receptor. FIGS. 1-4 show schematic diagrams of the interactions of Ex-4 and Ex-4iii with the receptor, with the C-terminal end of Ex-4iii forming new hydrogen bonds and hydrophobic interactions with the receptor, while the active alpha helix structure is more stable and the binding force of the receptor active domain is improved.
Example 3 Ai Saina peptide analog Activity to stimulate cAMP release from murine islet tumor cells RIN-m5F in vitro
(1) Test materials and related reagent formulations
PBS: 8g of sodium chloride, 0.2g of potassium chloride, 1.44g of disodium hydrogen phosphate and 0.24g of monopotassium phosphate are dissolved in water and diluted to 1000ml, the pH is adjusted to 7.2, the mixture is autoclaved at 121 ℃ for 15min, and the mixture is preserved at 4 ℃.
Ai Saina peptide Ex-4 and Ai Saina peptide analog Ex-4 (i-vi) polypeptide sample preparation: accurately weighing 10mg of polypeptide samples, accurately preparing a sample solution of 0.1mg/ml by using sterile water for injection, determining the content of the polypeptide by a nitrogen determination method, diluting the polypeptide according to the content of the polypeptide to 100ng/ml of polypeptide concentration, and diluting the polypeptide according to a ratio of 2 times to obtain 8 dilution sample solutions of 100ng/ml, 50ng/ml, 25ng/ml, 12.5ng/ml, 6.25ng/ml, 3.12ng/ml, 1.56ng/ml and 0.78ng/ml of polypeptide respectively.
(2) Test method
Taking RIN-m5F cells with good growth state, digesting, and counting 5×10 cells 5 ～8×10 5 Culturing for 18-36 h at 37 ℃ per mL; digestion with 0.25% pancreatin at 3.5X10 5 Inoculating 24-well cell plate at a volume of 0.5mL per well, placing at 37deg.C and 5% CO 2 Culturing for 24-36 hr. Discarding cell solution, adding cell maintenance solution (RPMI 1640 culture solution containing 5mg/mL bovine serum albumin 1.0 mL/well, 37 deg.C, 5% CO) 2 Culturing for 15min. The supernatant was discarded, and a cell maintenance solution containing 1mM IBMX, 0.9 mL/well, 37℃and 5% CO was added 2 Culturing for 15min. Adding 0.1 mL/Kong Daice sample as soon as possible, mixing with light shaking, and mixing at 37deg.C with 5% CO 2 Culturing for 15min. The cell culture plates were removed and placed on ice, the supernatant was discarded, and 1.0 mL/Kong Yuleng of PBS was added for washing twice, and the PBS was discarded. 300. Mu.L of cell lysate was added to each well, and the mixture was frozen and lysed twice at-80℃and 37℃for 30min each time. Taking out cell lysate, centrifuging at 12000r/min for 10min, referring to detection instruction book of cAMP-ELISA kit, taking supernatant for OD value determination, and making curve of OD value and sample concentration, and the result is shown in figure 5.
As can be seen from FIG. 5, the Ai Saina peptide analogs Ex-4 (i-vi) designed according to the present invention all exhibited enhanced activity. Ex-4 and Ex-4 (i-vi) stimulated cAMP releasing activity of RIN-m5F in mouse pancreatic islet tumor cells at different drug concentrations. In vitro biological activity increases in a dose-dependent manner. Ex-4ii, ex-4iii and Ex-4iv stimulate the release of cAMP from murine islet tumor cells RIN-m5F more active than Ex-4i, ex-4v and Ex-4 vi. ED50 s of Ex-4 and Ex-4 (i-vi) stimulated the release of cAMP by mouse islet tumor cells RIN-m5F were determined to be 0.080nmol/L, 0.072nmol/L, 0.067nmol/L, 0.061nmol/L, 0.063nmol/L, 0.073nmol/L, 0.075nmol/L, respectively, at the same concentration. Wherein Ex-4ii, ex-4iii and Ex-4iv are respectively improved by 16.2%, 23.8% and 21.2% compared with Ex-4.
EXAMPLE 4 in vivo hypoglycemic effect in db/db mice model for type II diabetes
SPF grade BKS. Cg-m+/+ Leprdb/J (db/db) spontaneous diabetic mice: 128 animals, 6-8 weeks old, male and female halves, were purchased from Beijing Fu Biotech Co., ltd. Production license number is SYXK (Beijing) 2009-0004, quality certificate number: 0172750.
SPF grade BKS. Cg-m+/+ Leprdb/J negative control mice (db/dm) were used for control, and were purchased from Beijing Fu Biotech Co., ltd, for 16 animals, 6-8 weeks old, male and female halves. Production license number is SYXK (Beijing) 2009-0004, quality certificate number: 0172750.
mouse SPF animal house feeding environmental conditions: class 10000 air cleanliness, 0.1-0.2 m/s air flow speed, 20-50 Pa ambient pressure difference, 20-25 deg.C humidity 40-70%, ammonia concentration less than or equal to 14mg/m 3 Ventilation is carried out for 10-20 times per hour, animal illuminance is 15-20 lux, working illuminance is 150-300 lux, noise is less than or equal to 60dB, day and night light and shade alternation time is 12h/12h, male and female are separately fed, 4 animals are fed in each cage, and after one week of adaptive feeding, the experiment is carried out.
The sex of 128 db/db spontaneous diabetes mice is divided into 8 groups according to the random layering of the blood sugar values which are determined in advance, namely (1) a model group; (2) Ex-4 group, 5 μg/kg; (3) Ex-4i group, 5 μg/kg; (4) Ex-4 group, 5 μg/kg; (5) Ex-4iii group, 5 μg/kg; (6) Ex-4iv set, 5. Mu.g/kg; (7) Ex-4v group, 5. Mu.g/kg; and (8) Ex-4vi group, 5 μg/kg. 16 animals in each group, and the male and female parts are half. (9) 16 db/dm mice were used as normal control mice.
The 9 groups of mice were subcutaneously administered 1 time, the control group and the model group were injected with 10mL/kg of physiological saline, the Ex-4 group was injected with the Ex-4 sample at 5. Mu.g/kg, and the sample group was injected with the Ex-4 (i-vi) sample at 5. Mu.g/kg.
After administration, all mice are sampled and blood glucose is detected on an empty stomach for 0.5-12 hours. The intensity and duration of hypoglycemic effects (m±sd, n=16) after single administration of Ex-4, ex-4 (i-vi) samples were determined. The measurement results are shown in Table 2.
TABLE 2 influence of Ai Saina peptide Ex-4 and its analogs Ex-4 (i-vi) on blood glucose levels in mice after fasting administration
*P<0.01vs negative control;
# P<model group 0.01vs
Experimental results show that compared with a model group, the blood sugar level (P < 0.01) can be obviously reduced after the Ex-4 and Ex-4 (i-vi) groups are subcutaneously injected for 0.5 h; the hypoglycemic effect of Ex-4 can be maintained for 8 hours after single administration, and the blood sugar concentration is obviously increased after 12 hours of administration. The hypoglycemic effect of Ex-4 (i-vi) was maintained for 12h in a single administration, and the blood glucose level of Ex-4 (i-vi) was still lower than that of the model group (P < 0.05) in a single administration for 12 h. Therefore, the six analogues of Ex-4 (i-vi) have the effect of reducing blood sugar, and have longer effect of reducing blood sugar compared with the Ex-4, and particularly the effect of reducing blood sugar of Ex-4ii, ex-4iii and Ex-4iv is more remarkable. Ex-4 (i-vi) has stronger activity than Ex-4, longer efficacy maintaining time and longer half-life.
Sequence listing
<110> Lunan pharmaceutical group Co., ltd
<120> Ai Saina peptide analogues
<160> 7
<170> SIPOSequenceListing 1.0
<210> 1
<211> 39
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 1
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Xaa Xaa
20 25 30
Xaa Xaa Xaa Pro Pro Pro Ser
35
<210> 2
<211> 39
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 2
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Gly Ser
20 25 30
Gly Gly Thr Gly Gly Ser Cys
35
<210> 3
<211> 39
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 3
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Gly
20 25 30
Gly Gly Thr Gly Gly Ser Cys
35
<210> 4
<211> 39
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 4
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Gly Gly Thr Gly Gly Ser Cys
35
<210> 5
<211> 39
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 5
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Thr Gly Gly Ser Cys
35
<210> 6
<211> 39
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 6
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Ala Thr Gly Gly Ser Cys
35
<210> 7
<211> 39
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 7
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30
Ser Gly Ala Gly Gly Ser Cys
35
Claims (4)
1. Ai Saina peptide analogues are characterized in that the amino acid sequence is shown as SEQ ID NO. 1:
His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-
Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Xaa37-Xaa38-Xaa39-Xaa40-
Xaa41-Xaa42-Xaa43-Xaa44-Xaa45，
wherein Xaa37 is Gly, xaa38 is Ser, xaa39 is Gly, xaa40 is Gly, xaa41 is Thr, xaa42 is Gly, xaa43 is Gly, xaa44 is Ser, xaa45 is Cys; namely Ai Saina peptide analogue Ex-4i, the amino acid sequence of which is shown as SEQ ID NO. 2;
wherein Xaa37 is Pro, xaa38 is Gly, xaa39 is Gly, xaa40 is Gly, xaa41 is Thr, xaa42 is Gly, xaa43 is Gly, xaa44 is Ser, xaa45 is Cys; namely Ai Saina peptide analogue Ex-4ii, the amino acid sequence of which is shown as SEQ ID NO. 3;
wherein Xaa37 is Pro, xaa38 is Ser, xaa39 is Gly, xaa40 is Gly, xaa41 is Thr, xaa42 is Gly, xaa43 is Gly, xaa44 is Ser, xaa45 is Cys; namely Ai Saina peptide analogue Ex-4iii, the amino acid sequence of which is shown as SEQ ID NO. 4;
wherein Xaa37 is Pro, xaa38 is Ser, xaa39 is Ser, xaa40 is Gly, xaa41 is Thr, xaa42 is Gly, xaa43 is Gly, xaa44 is Ser, xaa45 is Cys; namely Ai Saina peptide analogue Ex-4iv, the amino acid sequence of which is shown as SEQ ID NO. 5;
wherein Xaa37 is Pro, xaa38 is Ser, xaa39 is Ser, xaa40 is Ala, xaa41 is Thr, xaa42 is Gly, xaa43 is Gly, xaa44 is Ser, xaa45 is Cys; namely Ai Saina peptide analogue Ex-4v, the amino acid sequence of which is shown as SEQ ID NO. 6;
wherein Xaa37 is Pro, xaa38 is Ser, xaa39 is Ser, xaa40 is Gly, xaa41 is Ala, xaa42 is Gly, xaa43 is Gly, xaa44 is Ser, xaa45 is Cys; namely Ai Saina peptide analogue Ex-4vi, the amino acid sequence of which is shown as SEQ ID NO. 7.
2. The Ai Saina peptide analog according to claim 1, wherein the C-terminus of the peptide chain of the Ai Saina peptide analog Ex-4 (i-vi) is amidated.
3. Use of a Ai Saina peptide analogue as claimed in claim 1 or 2 in the manufacture of a medicament for the treatment of diabetes and obesity.
4. The use according to claim 3, wherein the medicament comprises a Ai Saina peptide analogue according to claim 1 or 2 and a pharmaceutically acceptable carrier.
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| CN1802386A (en) * | 2003-06-12 | 2006-07-12 | 伊莱利利公司 | GLP-1 analog fusion plroteins |
| WO2015142140A1 (en) * | 2014-03-21 | 2015-09-24 | 애니젠 주식회사 | Novel exenatide analogue and use thereof |
| CN105593242A (en) * | 2013-07-11 | 2016-05-18 | 加州生物医学研究所 | Metal-carbonaceous bruch and method for producing same |
| CN105753963A (en) * | 2016-04-13 | 2016-07-13 | 中国药科大学 | High-activity exendin-4 analogue and pharmaceutical application thereof |
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| CN100429227C (en) * | 2005-06-29 | 2008-10-29 | 常州制药厂有限公司 | Exendin 4 polypeptide segment |
| CN101993485B (en) * | 2009-08-20 | 2013-04-17 | 重庆富进生物医药有限公司 | Peptide analog homologous dimer capable of accelerating insulin secretion and application thereof |
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| CN1802386A (en) * | 2003-06-12 | 2006-07-12 | 伊莱利利公司 | GLP-1 analog fusion plroteins |
| CN105593242A (en) * | 2013-07-11 | 2016-05-18 | 加州生物医学研究所 | Metal-carbonaceous bruch and method for producing same |
| WO2015142140A1 (en) * | 2014-03-21 | 2015-09-24 | 애니젠 주식회사 | Novel exenatide analogue and use thereof |
| CN105753963A (en) * | 2016-04-13 | 2016-07-13 | 中国药科大学 | High-activity exendin-4 analogue and pharmaceutical application thereof |
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| Title |
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| 艾塞那肽类似物的制备及降糖活性;夏春光等;《中国医药工业杂志》;20130910(第09期);全文 * |
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