CN116693652B

CN116693652B - GLP-1/GIP receptor dual agonist derivative and preparation method and application thereof

Info

Publication number: CN116693652B
Application number: CN202310964890.7A
Authority: CN
Inventors: 曹海燕; 林兆生; 朱志伟; 张海波; 李吉玲; 刘洋
Original assignee: Jilin Huisheng Biopharmaceutical Co ltd; Beijing Huizhiheng Biological Technology Co Ltd
Current assignee: Jilin Huisheng Biopharmaceutical Co ltd; Beijing Huizhiheng Biological Technology Co Ltd
Priority date: 2023-08-02
Filing date: 2023-08-02
Publication date: 2024-01-05
Anticipated expiration: 2043-08-02
Also published as: CN116693652A

Abstract

The invention relates to the technical field of genetic engineering, in particular to a GLP-1/GIP receptor dual agonist derivative, a preparation method and application thereof. The GLP-1/GIP receptor dual-agonist derivative has more reasonable activity ratio of GLP-1 receptor agonist and GIP receptor agonist, has excellent hypoglycemic and weight-reducing effects, and can be used for treating metabolic diseases such as diabetes, obesity and the like.

Description

GLP-1/GIP receptor dual agonist derivative and preparation method and application thereof

Technical Field

The invention relates to the technical field of genetic engineering, in particular to a GLP-1/GIP receptor dual agonist derivative, a preparation method and application thereof.

Background

Diabetes is a group of metabolic disorders of carbohydrates, proteins, fats, etc. caused by absolute or relative hyposecretion of insulin and/or dysfunction of insulin utilization, and is mainly marked by hyperglycemia, and can be caused by various factors such as heredity and environment. Diabetes is one of the three major fatal diseases in humans, with mortality rates inferior to cardiovascular and cerebrovascular diseases and cancers.

Incretin (Incretin) is a factor that stimulates insulin secretion contained in crude secretin peptide products. There are two known incretins to date: glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP). GIP is a 42 amino acid gastrointestinal regulatory peptide secreted by K cells in the duodenum and upper jejunum. The earliest studies found that GIP can inhibit gastric acid secretion under certain conditions, and thus, it was first called "aprotinin", but the subsequent studies found that it had an effect of secreting insulin from pancreatic β cells and protecting pancreatic β cells by stimulating them in the presence of glucose, and had a physiological effect in glucose homeostasis; meanwhile, GIP induces glucagon production under hypoglycemic conditions, thereby maintaining blood glucose balance (Christensen et al. (2011) Diabetes 60:3103-9), which is then renamed as a glucose-dependent insulinotropic polypeptide.

GLP-1 is a glucagon secreted by intestinal L cells, has the same amino acid sequence as glucagon, has multiple functions of promoting insulin secretion and biosynthesis, inhibiting glucagon secretion, inhibiting gastric emptying and the like in a glucose-dependent manner, and simultaneously, GLP-1 is taken as an intestinal sex hormone which is released into blood only under the stimulation of nutrient substances (especially carbohydrate), has the function of promoting insulin secretion in a glucose concentration-dependent manner, can play a role in reducing blood glucose when blood glucose rises, inhibits glucagon secretion, increases satiety and reduces hunger sensation, thereby achieving the effect of reducing blood glucose. In addition, GLP-1 can also act on the central nervous system (especially hypothalamus) to inhibit appetite and reduce food intake, so that the human body can produce satiety and appetite reduction, and the intake of calories is reduced.

The discovery of incretins has successfully developed two new classes of drugs for the treatment of diabetes, GLP-1 receptor agonists that mimic the effects of endogenous GLP-1 and small molecule compounds that inhibit the enzymatic inactivation of both endogenous GLP-1 and GIP (oral DPP-4 inhibitors), respectively, GLP-1 receptor agonists that have been marketed today such as ByettaTM, bydureonTM, lixisenatideTM and VictozaTM, etc.; DPP-4 inhibitors such as JanuviaTM, galvusTM, onglyzaTM and Traj eta TM are now on the market. Unlike GLP-1, which was expected from its birth, GIP was found earlier than GLP-1, but it has not been a drug for treating diabetes, mainly because early studies did not see the insulin secretion promoting effect of GIP in type 2 diabetics. However, in recent years, the role of GIP is gradually demonstrated, making it one of the hot targets. Weidinmailer et al reported that DPP-4 resistant GIP analogs have anti-apoptotic effects (Weidinmailer, SD, PLOS One 5 (3): e9590 (2010)). Meanwhile, it is described in WO 2013/164483, WO 2014/192284 and WO 2011/119657 that certain GIP analogs exhibit both GIP and GLP-1 activity. Interestingly, in the mouse model of diabetes and obesity, the combination of the GLP-1 receptor agonist Liraglutide and the acylated GIP analog showed superior hypoglycemic (glucose-lower) and insulinotropic) effects (Gault, VA, clinical Science 121:107-117 (2011)) to treatment with Liraglutide and GIP analog alone. GIP analog ZP4165 has also been reported to enhance the hypoglycemic effect and slimming effect of GLP-1 agonists in Nbran rregaard, P.K., et al (2018) Diabetes Obes Metab 20:60-68). Finan, B.et al report a single molecule dual insulinotropic (unimolecular dual incretins) polypeptide with coactivation to GLP-1R and GIPR, which showed good insulin secretion and hypoglycemic effects in db/db mice, ZDF rats, monkeys and humans (Finan et al (2013) Sci Transl Med 5:2090151).

In view of the findings of GIP effects, the development of dual-target co-agonists capable of acting simultaneously at the GIP receptor and GLP-1 receptor is becoming a popular research direction. Compared with the composite medicine of a plurality of single receptor agonists, the single-molecule multiple receptor agonist can be activated simultaneously due to different signal paths, thereby having the advantages of maximized curative effect, reduced side effect, more stable pharmacokinetic property and the like. Some GIP analog-based GLP-1 and GIP receptor dual agonist derivative compounds are described, for example, in patents CN201380032714.4, CN201480060723.9, CN 20160005007. X, etc. To date, only the dual GLP-1 and GIP receptor agonist derivatives, telpoismin, are marketed in the United states as being offered by Gift corporation.

Thus, there is still a need to provide a variety of dual GLP-1 and GIP receptor agonists and derivatives thereof to provide superior hypoglycemic and weight-reducing efficacy.

Disclosure of Invention

In the present invention, the term "derivative" in relation to a peptide (e.g., GLP-1 or GIP) means a peptide or analog thereof that has been chemically modified (e.g., covalently modified, etc.). Typical modifications include amides, sugars, alkyl groups, acyl groups, esters, and the like.

As used herein, the term "dual GLP-1/GIP receptor agonist" is a class of compounds that agonize the dual incretin peptides of the human glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-l) receptor.

The term "inclusion-promoting sequence" refers to a polypeptide sequence that is linked prior to a protein or polypeptide of interest for promoting expression or formation of inclusion bodies.

The term "expression-promoting peptide" refers to a polypeptide sequence that is linked after a leader peptide and before a protein or polypeptide of interest, further enhances the expression of the protein or polypeptide of interest, or is capable of promoting the expression of the protein or polypeptide of interest that is difficult to express by conventional leader peptides.

The term "peptide" refers to a molecule comprising amino acid sequences linked by peptide bonds, whether in length, post-translational modification or function.

In the present invention, the term "pharmaceutically acceptable salt" refers to a salt of a polypeptide or protein that retains the biological activity of the parent.

In the present invention, the term "pharmaceutically acceptable adjuvant" broadly refers to any component other than the active therapeutic ingredient. The auxiliary materials can be inert substances, inactive substances and/or non-pharmaceutically active substances.

In the present invention, the term "pharmaceutically acceptable carrier" includes any standard pharmaceutical carrier, e.g., phosphate buffered saline solution, water, emulsions, such as oil/water or water/oil emulsions, and various types of wetting agents.

In the present invention, the term "composition/formulation" may be a stabilized formulation. The term "stabilized formulation" refers to a formulation having improved physical and/or chemical stability (preferably in combination). In general, until the expiration time is reached, the formulation must be stable during use and storage (meeting recommended use and storage conditions).

In the present invention, the term "vector" refers to a vector into which a nucleotide fragment encoding a protein or polypeptide can be operably inserted to cause expression of the protein or polypeptide. Vectors may be used to transform, transduce or transfect host cells such that they express the carried genetic element within the host cells. Examples of vectors include plasmids, artificial chromosomes, phages, viral particles and the like. The vector may contain a variety of elements for controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. The vector may also include materials that facilitate its entry into the cell, including but not limited to viral particles, liposomes, or protein envelopes.

The vector may be a recombinant expression vector or a cloning vector. The present invention provides vectors (e.g., expression vectors) comprising a nucleic acid sequence encoding a GIP and GLP-1 dual target polypeptide of the invention. Examples of vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex viruses), poxviruses, baculoviruses, papillomaviruses, papovaviruses (e.g., SV 40), lambda and M13 phages, plasmids such as pcDNA3.3, pMD18-T, pOptivec, pCMV, pEGFP, pIRES, pQD-Hyg-GSeu, pALTER, pBAD, pcDNA, pCal, pL, pET, pGEMEX, pGEX, pCI, pEGFT, pSV2, pFUSE, pVITRO, pVIVO, pMAL, pMONO, pSELECT, pUNO, pDUO, psg5L, pBABE, pWPXL, pBI, p15TV-L, pPro18, pTD, pRS10, pLexA, pACT2.2, pCMV-SCRIPT.RTM, pCDM8, pCDNA1.1/amp, pcDNA3.1, pRc/RSV, PCR2.1, pEF-1, pFB, pSG5, pXT1, pCDEF3, pSVSPORT, pEF-Bos, and the like.

In the present invention, the term "recombinant expression vector" is a nucleic acid molecule encoding a gene which is expressed in a host cell and which contains the necessary elements to control the expression of the gene. Typically, the expression vector comprises a transcription promoter, a gene of interest, and a transcription terminator.

In the present invention, a host cell refers to a cell into which a vector comprising a nucleotide sequence fragment encoding a protein or polypeptide of interest can be introduced for cloning or gene expression. Host cells suitable for cloning or expressing the DNA in the vectors herein are prokaryotes, yeast, or higher eukaryote cells.

In the present invention, the term "treating" includes inhibiting, slowing, stopping or reversing the progression or severity of an existing symptom or condition.

In the present invention, the term "Tirzepatide" refers to a GLP-1/GIP receptor co-agonist polypeptide having the peptide backbone and the overall compound structure of CAS registry No. 2023788-19-2.

Tirzepatide (telpopeptide) is a GLP-1 and GIP receptor dual-agonism polypeptide derivative developed by Gift corporation of America, and is the only GLP-1 and GIP receptor dual-agonism polypeptide derivative currently marketed in bulk. Tirzepatide can modulate beta cell function through different signal transduction pathways, activate adenylate cyclase, increase intracellular concentrations of cyclic adenosine monophosphate (cAMP), and thereby activate protein kinase A and cAMP direct exchanger protein (Epac). Existing studies have demonstrated that there are two subtypes of Epac, of which type 1 may have a protective effect on beta cells, while type 2 promotes glucose-induced insulin secretion. Also, researchers believe that this may be one of the reasons that Tirzepatide has a better hypoglycemic effect compared to GLP-1 receptor agonists. In conclusion, tirzepatide is not simply added with GLP-1 receptor agonist and GIP receptor agonist, but complements through the mechanism of the two, realizes the synergistic force-gathering effect of '1+1 > 2', and further improves the curative effect and safety of the medicine.

From the current data, tirzepatide has a hypoglycemic and weight-reducing effect which is not inferior to that of cord Ma Lutai, even superior to the clinical effect of the latter. However, the clinical dosage of Tirzepatide is several times (5 times to more than ten times) that of the cable Ma Lutai, and the dosage of Tirzepatide is Gao Yusuo Ma Lutai injection.

In order to solve the technical problems, the invention provides a GLP-1/GIP receptor dual agonist derivative, wherein the polypeptide sequence of the GLP-1/GIP receptor dual agonist derivative has a higher humanized sequence, and has relatively high GLP-1 receptor agonist activity and relatively low GIP receptor agonist activity. Not only has excellent blood sugar reducing effect, but also has the effects of reducing appetite and weight of patients, and can solve the problem that the traditional insulin and the derivatives thereof can cause the weight increase of the patients. Preferably, the GLP-1/GIP receptor dual agonist derivatives of the invention have a polypeptide structure having an amino acid sequence of the formula:

X-Aib-EGTFTSDYSILLDQIAQKDFVQWLIEGGPSSG；

wherein X represents His or Tyr.

Further preferably, the lysine of the dual GLP-1/GIP receptor agonist derivative is linked with a fatty acid side chain to prolong the action time and achieve a long-acting effect.

As a further preferred embodiment of the present invention, the fatty acid side chain is HOOC- (CH) ₂ ) _14~20 CO-, preferably COOH (CH) ₂ ) _18~20 CO-; more preferably HOOC- (CH) ₂ ) ₁₆ -CO-or COOH (CH ₂ ) ₁₈ CO-. The side chain is linked to the epsilon amino group of lysine via a linker.

Further preferably, the linker is selected from one of divalent groups represented by formula 1 to formula 12:

（1）、/>（2）、/>（3）、

（4）、

（5）、

（6）、

（7）、/>（8）、

（9）、

（10）、

（11）、

（12）；

where m is an integer of 0 to 6, such as 0, 1, 2, 3, 4, 5, 6, etc., n is an integer of 1 to 3, such as 1, 2, 3, etc., s is an integer of 0 to 3, such as 0, 1, 2, 3, etc., t is an integer of 0 to 4, such as 0, 1, 2, 3, 4, etc., and p is an integer of 1 to 23, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, etc.

More preferably, the linker is selected from divalent groups represented by the following structural formula:

；

wherein s is 1, n is 1 or 2.

For the preferred linker moiety described above (when n is 1), it may be denoted gamma-Glu-OEG-OEG according to IUPAC nomenclature; wherein OEG is "2- [2- (2-aminoethoxy)) Ethoxy group]The abbreviation of acetyl ". Selecting HOOC (CH) ₂ ) ₁₆ When CO-is used as the side chain, the combination of the side chain and the linker (acyl group) may be referred to as "[2- (2- [2- (2-) 4- (17-carboxyheptadecanoyl amide) -4 ]S) -carboxybutyrylamino group]Ethoxy) ethoxy]Acetamido) ethoxy]Ethoxy) acetyl]”。

In a second aspect, the invention also provides a soluble pharmaceutical composition comprising a GLP-1/GIP receptor dual agonist derivative or a pharmaceutically acceptable salt thereof. The pharmaceutical composition also comprises pharmaceutically acceptable auxiliary materials.

The invention also provides a preparation method of the GLP-1/GIP receptor dual agonist derivative, which can adopt a chemical synthesis method or a fermentation expression method for preparing recombinant genetically engineered bacteria.

Specifically, the third aspect of the invention provides a fusion protein which can be obtained by fermenting and expressing recombinant genetically engineered bacteria and is used for further obtaining the GLP-1/GIP receptor dual agonist derivative of the first aspect of the invention. Specifically, the fusion protein is formed by sequentially connecting at least an inclusion body promoting sequence, an expression promoting peptide sequence, an enzyme cleavage site sequence and a GLP-1/GIP receptor dual agonist polypeptide precursor. Wherein the amino acid sequence of the GLP-1/GIP receptor dual agonist polypeptide precursor sequence is shown as SEQ ID NO. 1, the amino acid sequence of the inclusion body promoting sequence is shown as SEQ ID NO. 2, the amino acid sequence of the expression promoting peptide sequence is shown as SEQ ID NO. 3, and the amino acid sequence of the cleavage site sequence is shown as SEQ ID NO. 4. The fusion protein has the technical effects of promoting expression and improving yield.

As a preferred embodiment of the invention, the amino acid sequence of the fusion protein is selected from SEQ ID NO.5. The specific sequences are shown in Table 1.

TABLE 1

In a fourth aspect, the invention provides a polynucleotide fragment for encoding the fusion protein, wherein the nucleotide sequence of the polynucleotide fragment is selected from SEQ ID NO.6.

SEQ ID NO.6：

atgtttaaatttgaatttaaatttgaggaaggcacctttacgagcgacgtgagcagctatctggaaggccaagcggcgaaagaatttattgcgtggctggtgcgcggccgcggcgatgacgatgataaagagggcacctttacgagcgattatagcattctgctggatcagattgcgcagaaagattttgtgcagtggctgattgaaggcggcccgagcagcggc

In a fifth aspect the invention provides an expression vector comprising a polynucleotide fragment according to the fourth aspect of the invention. The expression vector can be selected from recombinant pET-28a (+) expression vector, recombinant pET-30a (+) expression vector or recombinant pET-32a (+) expression vector, and preferably recombinant pET-30a (+) expression vector.

The sixth aspect of the present invention provides a recombinant escherichia coli engineering bacterium, which comprises the expression vector provided in the fifth aspect, specifically, escherichia coli can be selected from BL21 (DE 3), and a specific construction method can be adopted:

s1, synthesizing a polynucleotide fragment, wherein the polynucleotide fragment is specifically shown as SEQ ID NO. 6;

s2, cloning the polynucleotide fragment into a plasmid pET-30a (+) to construct an expression vector;

s3, transforming the expression vector into escherichia coli BL21 (DE 3) to obtain recombinant escherichia coli engineering bacteria.

The seventh aspect of the present invention provides a preparation method of a dual GLP-1/GIP receptor agonist derivative, at least comprising the following steps:

s4, culturing the recombinant escherichia coli engineering bacteria to express fusion proteins;

s5, carrying out denaturation, renaturation and enzyme digestion on the fusion protein to obtain a GLP-1/GIP receptor dual agonist polypeptide precursor;

s6, connecting a fatty acid side chain on the GLP-1/GIP receptor dual agonist polypeptide precursor to obtain a fatty acid modified derivative precursor; the fatty acid side chain is HOOC- (CH) ₂ ) _14~20 -CO-, the side chain being linked to the epsilon amino group of lysine by a linker-gamma-Glu-AEEa-eea-;

s7, modifying the N end of the derivative precursor after fatty acid modification by a dipeptide group, wherein the dipeptide group is selected from His-Aib-or Tyr-Aib-; obtaining the GLP-1/GIP receptor dual agonist derivative.

Specifically, S4 in the above preparation method includes:

s41, activating strains to obtain an activated seed culture solution;

s42, fermentation culture: inoculating the activated seed culture solution to a fermentation medium;

s43, induction expression: when the OD600 value is 155-165, IPTG is added for induction expression, and the final concentration of the IPTG is 0.9-1.1 mmol.

Specifically, S6 in the above preparation method includes:

preparing a solution of a GLP-1/GIP receptor dual agonist polypeptide precursor and a fatty acid side chain precursor compound, respectively, and mixing the GLP-1/GIP receptor dual agonist polypeptide precursor and the fatty acid side chain precursor compound in a molar ratio of 1:3 to 5, and preferably in a molar ratio of 1:4, mixing. Specifically, the concentration of GLP-1/GIP receptor dual agonist polypeptide precursor was quantified by HPLC, and the amount of fatty acid side chain precursor compound used was calculated.

Further preferably, the fatty acid side chain precursor compound may employ:

tBuO-Ste-Glu(AEEA-AEEA-OH)OtBu。

specifically, water is used as a solvent to prepare a solution of a GLP-1/GIP receptor dual agonist polypeptide precursor, the concentration is 4-6 mg/mL, and a pH regulator is adopted to regulate the pH to 11.0-11.5; organic solvents are used to prepare solutions of fatty acid side chain precursor compounds. The organic solvent can be selected from alcohols organic solvent and nitriles organic solvent, and acetonitrile is preferred. The pH regulator is alkaline solvent, and sodium hydroxide solution, preferably sodium hydroxide solution 1M, can be used.

Specifically, S7 in the above preparation method includes: the solution of the dipeptide group compound is prepared by adopting an organic solvent, wherein the organic solvent can be selected from alcohol organic solvents and nitrile organic solvents, and acetonitrile is preferred. The dipeptide based compound may be selected from Boc-His (Trt) -Aib-OH and Boc-Tyr (tBu) -Aib-OH. Mixing fatty acid modified derivative precursor with dipeptide group compound in a molar ratio of 1:2 to 4, preferably in a molar ratio of 1:3, mixing. Specifically, the concentration of the fatty acid modified derivative precursor was quantified by HPLC, and the amount of the dipeptide based compound used was calculated.

After the reaction is finished, stopping the reaction, and separating, deprotecting and purifying to obtain the GLP-1/GIP receptor dual agonist derivative.

Specifically, the reaction is stopped by diluting and adjusting the pH to be acidic, specifically, water is added to dilute the reaction system to 4-8 times, preferably 5 times, and then a pH regulator is added to adjust the pH to 4.5-5.0. The pH adjustor adopts an acidic solution, such as a 1M citric acid solution or a 10% acetic acid solution.

The specific deprotection method is selected based on the protecting groups employed in the fatty acid side chain precursor compound, the dipeptide group compound.

The purification can be carried out by a protein purification instrument, polystyrene filler is adopted, and 0-100% eluent (containing 5-20 mM ammonium acetate and 70-90% acetonitrile) is used for gradient elution. Specifically, elution may be performed at a ratio of 0, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% in order.

The eighth aspect of the invention provides application of the GLP-1/GIP receptor dual agonist derivative or pharmaceutically acceptable salt thereof or the pharmaceutical composition in preparing medicaments for treating metabolic diseases or reducing weight. In particular, metabolic disorders include, but are not limited to: diabetes (type I diabetes, type II diabetes), overweight and obesity, steatohepatitis (NASH, ASH), cardiovascular disease, fatty liver, cirrhosis, nonalcoholic fatty liver disease, metabolic syndrome, and various diabetic complications.

Compared with the prior art, the technical scheme provided by the invention has the following advantages:

the GLP-1 and GIP receptor co-excitation coding gene sequence provided by the invention can be used for treating insulin-dependent diabetes mellitus, and has more reasonable GLP-1 and GIP activity ratio.

The GLP-1/GIP receptor dual agonist derivative provided by the invention can be used for treating metabolic diseases such as diabetes, obesity and the like, and has excellent hypoglycemic and weight-reducing effects.

Drawings

FIG. 1 is a graph showing the statistical result of weight change of mice;

FIG. 2 is a graph showing the statistical result of the weight change rate of mice;

FIG. 3 is a graph showing the cumulative food intake statistics of mice:

FIG. 4 is a statistical graph of the blood glucose reduction results of mice.

Detailed Description

In order that the above objects, features and advantages of the invention will be more clearly understood, a further description of the invention will be made. It should be noted that, without conflict, the embodiments of the present invention and features in the embodiments may be combined with each other.

In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, but the present invention may be practiced otherwise than as described herein; it will be apparent that the embodiments in the specification are only some, but not all, embodiments of the invention.

Example 1:

this example is presented to illustrate the procedure for the preparation of GLP-1/GIP receptor dual agonist derivatives using recombinant expression and chemical modification:

(1) Construction of an expression plasmid:

sequentially concatenating inclusion body promoting sequence (SEQ ID NO: 2), expression promoting peptide sequence (SEQ ID NO: 3), EK digestion sequence (SEQ ID NO: 4) and GLP-1/GIP receptor dual agonist polypeptide precursor sequence (SEQ ID NO: 1) to obtain fusion protein sequence (SEQ ID NO: 5):

MFKFEFKFEEGTFTSDVSSYLEGQAAKEFIAWLVRGRGDDDDKEGTFTSDYSILLDQIAQKDFVQWLIE GGPSSG

the coding gene sequence of the fusion protein is constructed, and a chemical synthesis mode is used for obtaining a gene fragment (SEQ ID NO: 6):

the above fragment was inserted into the prokaryotic expression plasmid pET-30a (+) through NdeI and XhoI sites and verified by sequencing. The resulting expression plasmid for transformation assays was designated pET-30a (+) -HSP005-012.

(2) Construction of recombinant engineering bacteria expressing the GLP-1/GIP receptor dual agonist polypeptide precursor:

BL21 competent cells (TransGenBiotech.) were thawed (50. Mu.L) on ice, the corresponding expression plasmid constructed was added, gently shaken and left in ice for 30 minutes. The centrifuge tube was then rapidly transferred to an ice bath for 2 minutes without shaking the tube after a subsequent heat shock in a 42℃water bath for 30 seconds. 500. Mu.L of sterile LB medium (without antibiotics) was added to the centrifuge tube, and after mixing, the mixture was incubated at 37℃for 1 hour at 180 rpm to resuscitate the bacteria. mu.L of transformed competent cells were aspirated and plated on LB agar medium plates containing kanamycin resistance, and the cells were spread evenly. The plate was placed at 37℃until the liquid was absorbed, the plate was inverted, and incubated overnight at 37 ℃. The next day, monoclonal colonies in the transformation plates were picked using an inoculating loop, inoculated in 15 mL sterile LB medium (containing antibiotics), cultured overnight at 30℃and deposited as primary seed solution of the recombinant engineering bacteria.

(3) Fermenting and expressing recombinant engineering bacteria:

1) Adding 50 μl of the first seed liquid bacterial liquid into 2×LB culture medium of 50 mL, adding 50 μl of kanamycin, mixing, standing in a constant temperature shaker, and culturing at 37deg.C and 200 rpm overnight with OD600 > 5.0;

2) The overnight cultured primary seeds 40 and mL in step 1) were inoculated into 200 mL of 2 XLB medium, and 200. Mu.L of kanamycin was added. Mixing, placing in a constant temperature oscillator, and culturing at 37deg.C and 200 rpm for 3 hr to obtain secondary seed culture solution with OD600 of more than 3.0.

3) Taking the secondary seeds 60 and mL in the step 2), and mixing according to the weight ratio of 1:10 (600 mL) in a fermentation medium, culturing in a 2L fermentation tank, when the OD600 value of the culture broth is detected to be about 160, starting to access IPTG, and culturing 24 h after induction to finish culturing, and centrifuging in a tank; the expressed bacterial liquid was centrifuged at 8000 g for 30 min. The yield of the thalli is about 300 g bacteria/L fermentation liquor, and the thalli obtained by centrifugation is delivered to an analysis department for measuring the expression quantity of the target protein, wherein the expression quantity is not less than 5 g/L.

(4) Purification of GLP-1/GIP receptor dual agonist polypeptide precursors:

100 g cell plasma was weighed and resuspended in 500 mL of 50 mM disodium hydrogen phosphate, pH8.0, 50 mM sodium chloride and sonicated in an ultrasonic cell disrupter for 30 min to disrupt cells. The homogenate is centrifuged at 13000 and g for 30 min at 4 ℃, and after centrifugation, the sediment is collected and dissolved by 8M urea, thus obtaining the sample before enzyme digestion. The resulting supernatant was concentrated by UniPS 30-300 (available from su-na micro-tech ltd) previously equilibrated with 10 mM ammonium acetate, 20% acetonitrile (equilibration solution 3). And (3) eluting the solution by using the balance solution 3, and then performing gradient elution according to 0-100% of eluent (10 mM ammonium acetate and 80% acetonitrile). The purity of the intermediate product of the GLP-1/GIP receptor dual agonist polypeptide precursor generated by the above purification process is higher than 70% by SDS-PAGE analysis.

Tag sequence excision using EK enzyme: the intermediate was diluted three-fold by adding 20 mM PB, pH7.4 buffer, and the mixture was subjected to EK enzyme treatment at 20 ℃): the intermediate product is 1:15 adding EK enzyme, mixing, and enzyme cutting overnight. The cleavage efficiency was approximately 80% by SDS-PAGE analysis.

Refinement of the GLP-1/GIP receptor dual agonist polypeptide precursor: uniPS 30-300 (available from Sony micro technology Co., ltd.) equilibrated with 10. 10 mM ammonium acetate, 20% acetonitrile (equilibration solution 3) was concentrated. After the equilibrium solution 3 is leached, the solution is eluted by 0 to 100 percent of eluent (10 mM ammonium acetate and 80 percent acetonitrile) in gradient, and the purity is about 90 percent by SDS-PAGE. And adding 0.2M disodium hydrogen phosphate into the eluted sample to ensure that the final concentration is 20 mM, and regulating the pH value to 4.8-5.0,4 ℃ by using 1M citric acid to perform acid precipitation overnight. SDS-PAGE detection yield is more than 90%. Centrifuging at 13000 and g at 4deg.C for 30 min, collecting precipitate, and storing at-20deg.C.

(5) Preparation of GLP-1/GIP receptor dual agonist derivatives:

fatty acid modification: adding water into the GLP-1/GIP receptor dual agonist polypeptide precursor precipitate prepared and collected in the embodiment, preparing into 5 mg/mL solution, adding 1M sodium hydroxide to adjust the pH to 11.0-11.5, shaking uniformly to completely dissolve the protein, and quantifying the concentration of the GLP-1/GIP receptor dual agonist polypeptide precursor by HPLC. In a molar ratio of GLP-1/GIP receptor dual agonist polypeptide precursor to tBuO-Ste-Glu (AEEA-AEEA-OH) OtBu (CAS: 1118767-16-0) of 1:4 weighing fatty acid powder and dissolving in acetonitrile. Mixing the GLP-1/GIP receptor dual agonist polypeptide precursor sample with a fatty acid solution to obtain a fatty acid modified derivative precursor, wherein the mixed solution is used for the subsequent steps. Fatty acid modification is linked with tBuO-Ste-Glu (AEEA-AEEA-OH) OtBu to the epsilon amino group of lysine 18 of the GLP-1/GIP receptor dual agonist polypeptide precursor.

Dipeptide modification: and adding 1M hydrochloric acid into the fatty acid modified derivative precursor to adjust the pH to 9.0-9.5, then adding a dipeptide group compound, and quantifying the concentration of the precursor B by HPLC. The molar ratio of the fatty acid modified derivative precursor to the dipeptide based compound is 1:3 weighing dipeptide group compound powder and dissolving in acetonitrile. The dipeptide based compound is selected from Boc-His (Trt) -Aib-OH or Boc-Tyr (tBu) -Aib-OH.

After the reaction, the mixture after the reaction is diluted by 5 times by adding water, the pH is regulated to 4.8 by using 1M citric acid (or 10% acetic acid) to stop the reaction, the reaction is placed at the temperature of 4 ℃ for standing and acid precipitation for 10 min, after the acid precipitation, 13000 g is centrifugated for 30 min at the temperature of 4 ℃, and the precipitate is placed at the temperature of minus 80 ℃ for preservation. The dipeptide group is linked to the epsilon amino group of glutamic acid at position 1 of the GLP-1/GIP receptor dual agonist polypeptide.

Deprotection and purification of fatty acids and dipeptides: and adding trifluoroacetic acid into the acid precipitation sample until the final concentration of the polypeptide is about 10 mg/mL, oscillating to dissolve the precipitate, standing at room temperature for deprotection for 30 min, and dripping 4M sodium hydroxide into the reaction solution to adjust the pH to 7.5-8.5 to terminate the reaction. The reaction solution after termination was concentrated by a protein purification chromatography system (Saikovia SDL 100) at a flow rate of 4 mL/min by pumping UniPS 10-300 (available from Sonchi micro technologies Co., ltd.) previously equilibrated with 10 mM ammonium acetate, 20% acetonitrile (equilibration solution 3). And (3) eluting the balance liquid 3, then carrying out gradient elution according to 0-100% eluent (10 mM ammonium acetate and 80% acetonitrile), and collecting an eluting peak, wherein the purity of the eluting peak is about 90% through RP-HPLC detection.

Diluting the eluting peak with water for 3 times, and acid precipitating to adjust pH to 4.80,4 deg.C for 30 min. Adding PBS buffer solution (pH 7.0) into the precipitate after centrifugation, re-dissolving, and freezing at-80 ℃. The GLP-1/GIP receptor double agonist derivative is named HSP005-012-C18.

According to the method of this example, tBu-OOC- (CH) ₂ ) ₁₈ -CO-Glu (AEEA-AEEA) -OtBu replaces the tBuO-Ste-Glu (AEEA-AEEA-OH) OtBu described above to prepare a GLP-1/GIP receptor dual agonist derivative with eicosane side chain designated HSP005-012-C20.

Example 2

This example was used to determine the cellular activity of the GLP-1/GIP receptor dual agonist derivatives prepared in example 1:

assay for GLP-1 receptor binding Activity

To determine the in vitro GLP-1 bioactivity of the samples prepared in example 1, a Fire-LumitM luciferase assay was performed in HEK293/Luc/GLP-1R cells stably expressing human GLP-1 receptor to determine the response of intracellular cyclic adenosine monophosphate (cAMP).

HEK293/Luc/GLP-1R cells in the logarithmic growth phase were selected, and after trypsin digestion, they were washed once with DPBS. Cells were resuspended in Dulbecco's Modified Eagle Medium (DMEM) medium, stained with 0.2% trypan blue, and counted. Cell density was adjusted to 8.0X10 with DMEM ⁵ mu.L of each well was inoculated into 96-well cell plates at 37℃in 5% CO ₂ Culturing overnight in an incubator. The control tirzepatide and the HSP005-012-C18 or HSP005-012-C20 samples prepared in example 1 of the present invention were serially diluted 5-fold from 320 nM to 0.004 nM, respectively, in DMEM medium. The prepared control and sample were added to the cell culture plate at 50. Mu.L/well, respectively, and at 37℃with 5% CO ₂ Reaction 6 h in incubator. Taking out the cell culture plate, balancing to room temperature, adding 100 mu L of Fire-Lumi detection solution into each hole, uniformly mixing, reacting at room temperature for 5min, and detecting fluorescence intensity in a multifunctional enzyme-labeling instrument.

The concentration that caused 50% activation of the maximum response (EC 50) was calculated using a four parameter regression calculation. The results are shown in tables 2 and 3.

TABLE 2

TABLE 3 Table 3

(two) GIP receptor binding Activity assay

To determine the in vitro GIP biological activity effect prepared in example 1, the response of intracellular cyclic adenosine monophosphate (cAMP) was determined using homogeneous time resolved fluorescence (HTRF, cisbio) in a CHO K1 GIPR cell line overexpressing the human GIP receptor. The method comprises the following specific steps:

CHO-K1/GIPR cells in log phase were selected, trypsinized and washed once with DPBS. Cells were resuspended in working solution (Ham's F-12K medium with 0.5 mM IBMX), stained with 0.2% trypan blue, and counted. Cell density was adjusted to 8.0X10 ⁵ mu.L/mL, 5. Mu.L/well, was seeded into 96-well cell plates. 4-fold serial dilutions from 100 nM to 0.006 nM of control Tirzepatide were performed with working fluid; the samples of HSP005-012-C18 or HSP005-012-C20 of the present invention were serially diluted from 400 nM to 0.024-nM. The prepared control and sample were added to a cell culture plate in an amount of 5. Mu.L/well, respectively, and a 96-well plate was covered with a gas permeable membrane and placed at 37℃in 5% CO ₂ Incubate in incubator for 30 min. Taking out the 96-well plate, adding 5 mu L of cAMP d2 working solution into each well, uniformly mixing, adding 5 mu L cAMP Eu Cryptate antibody working solution into each well, uniformly mixing again, covering by a sealing film, incubating for 1 h at room temperature in a dark place, and reading the fluorescence ratio at 665/620 nm by using a multifunctional enzyme-labeled instrument.

The concentration that caused 50% activation of the maximum response (EC 50) was calculated using a four parameter regression calculation. The results are shown in tables 4 and 5.

TABLE 4 Table 4

TABLE 5

Example 3

This example was used to determine the efficacy of the GLP-1/GIP receptor dual agonist derivative prepared in example 1 against weight loss and feed intake inhibition in model mice:

(1) Experimental method

DIO model mice: and (3) purchasing 18C 57 mice with the age of 14-16 weeks, which are fed with high-fat feed (60% fat) by healthy SPF level, and carrying out random grouping after the healthy SPF level is fed for 5-7 days in an adaptive mode, wherein the weight of the C57 mice is 35-40 g. Each group contained 6 mice, grouped according to body weight, into G1 model control group, G2 positive control Tirzepatide (0.15 mpk) group, G3 HSP005-012-C18 (0.15 mpk) group. The first DAY of the formal dosing is denoted DAY1, dosing is performed every two DAYs, mice are weighed and food intake monitored each time, and DAY11 is not dosed and only weighing and food intake monitoring is performed.

Monitoring food intake: the food intake was monitored at each administration and 48 h after the last administration;

weight of: the 48 h mice were weighed at each dose and after the last dose;

the experimental results obtained are shown in table 6:

TABLE 6

Statistical analysis of all data from this experiment was performed using SPSS software. All values are expressed as mean ± standard deviation (mean ± SD). For normal distribution data, one-way ANOVA (one-way ANOVA) was used to compare group differences. For non-normal distribution data, the differences between groups were analyzed using either the Kruskal-Wallis H test or the Mann-Whitney U test. Correlation analysis uses the Spearman test. For all analyses, P <0.05 was considered statistically significant.

(2) Experimental results

Table 7 and fig. 1 are the results of the weight change statistics of the mice, and table 8 and fig. 2 are the results of the weight change rate statistics of the mice.

The calculation formula of the weight change rate is as follows: delta% = (Dayn-Day 1)/Day 1;

TABLE 7 statistical results of body weight changes (g)

Table 8: statistical results of weight change rate

From the results, it was found that the activity of the HSP005-012-C18 derivative against the positive control drug for weight loss was substantially the same at a 0.15 mpk drug concentration (30 nM) administered once every two days under test conditions of 5 administrations.

Table 9 is a statistical table of the intake, cumulative intake, and cumulative intake inhibition ratio, and the cumulative intake statistical chart is shown in FIG. 3:

table 9: statistical table of intake, accumulated intake and accumulated intake inhibition rate

From the results, the food intake inhibition rate of the HSP005-012-C18 derivative was superior to that of Tirzepatide, the positive control drug.

Example 4:

this example was used to determine the hypoglycemic effect of the GLP-1/GIP receptor dual agonist derivative prepared in example 1 against mice:

(1) Experimental method

SPF-class 7-8 week BKS-LeprRem/Gpt mice were selected, the mice which did not meet the experimental conditions were eliminated by randomly grouping according to body weight and blood sugar, and the other mice were randomly divided into 3 groups, each group comprising 8 experimental mice, which were blank model control group, positive control Tirzepatide (0.15 mpk), HSP005-012-C18 (0.15 mpk) group, respectively. Wherein the blank model control group was subcutaneously injected with blank solvent. The first Day of formal administration was noted Day1, and administration was 1 time. The dosage and the condition of the drug are shown in Table 10.

Blood glucose value: as shown in the experimental methods, the blood glucose readings of mice were obtained by a glucometer before the first administration (0 h) and 2 h, 4 h, 6 h, 8 h, 10 h, 24 h, 30 h, 48 h after the administration, and the blood glucose change curve was drawn to calculate the AUC area.

Table 10: administration mode

Statistical analysis of all data from this experiment was performed using SPSS software. All values are expressed as mean ± standard deviation (mean ± SD). For normal distribution data, one-way ANOVA (one-way ANOVA) was used to compare group differences. For all analyses, P <0.05 was considered statistically significant.

(1) Experimental results

The statistical results of the mice blood glucose reduction experiments are shown in Table 11, and the statistical graph of the results of the mice blood glucose reduction experiments is shown in FIG. 4.

Table 11-1: blood glucose reducing result statistical table for mice

Table 11-2: blood glucose reducing result statistical table for mice

* p <0.05 vs G1 blank; * P <0.01 vs G1 blank.

From the results, it was found that the hypoglycemic activity of the 0.15 mpk-administered group of the HSP005-012-C18 derivative was superior to that of Tirzepatide (same administration concentration).

The same experiment as in examples 3 and 4 above was conducted on HSP005-012-C20, and the experimental result showed that it also had a weight-reducing effect similar to Tirzepatide and a hypoglycemic activity superior to the latter.

The foregoing is only a specific embodiment of the invention to enable those skilled in the art to understand or practice the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown and described herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A GLP-1/GIP receptor dual agonist derivative or a pharmaceutically acceptable salt thereof, characterized in that the amino acid structure of the GLP-1/GIP receptor dual agonist derivative is as follows:

X-Aib-EGTFTSDYSILLDQIAQKDFVQWLIEGGPSSG；

wherein X represents His or Tyr;

the lysine of the GLP-1/GIP receptor dual agonist derivative is connected with a fatty acid side chain, and the fatty acid side chain is HOOC- (CH) ₂ ) _14~20 -CO-, said side chain being attached to the epsilon amino group of lysine by a linker;

the linker is selected from divalent groups represented by the following structural formula:

；

wherein s is 1 and n is 1.

2. The GLP-1/GIP receptor dual agonist derivative or a pharmaceutically acceptable salt thereof according to claim 1, wherein the fatty acid side chain is HOOC- (CH) ₂ ) ₁₆ -CO-or HOOC- (CH) ₂ ) ₁₈ -CO-。

3. A soluble pharmaceutical composition comprising the GLP-1/GIP receptor dual agonist derivative or a pharmaceutically acceptable salt thereof according to claim 1 or 2, wherein the soluble pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

4. The fusion protein is characterized by being formed by sequentially connecting an inclusion body promoting sequence, an expression promoting peptide sequence, an enzyme cleavage site sequence and a GLP-1/GIP receptor dual agonist polypeptide precursor sequence; the amino acid sequence of the GLP-1/GIP receptor dual agonist polypeptide precursor sequence is shown as SEQ ID NO. 1, the amino acid sequence of the inclusion body promoting sequence is shown as SEQ ID NO. 2, the amino acid sequence of the expression promoting peptide sequence is shown as SEQ ID NO. 3, and the amino acid sequence of the cleavage site sequence is shown as SEQ ID NO. 4.

5. The fusion protein of claim 4, wherein the amino acid sequence of the fusion protein is shown in SEQ ID NO.5.

6. A polynucleotide fragment for encoding the fusion protein of claim 4 or 5, wherein the nucleotide sequence of the polynucleotide fragment is selected from the group consisting of SEQ ID No.6.

7. An expression vector comprising the polynucleotide fragment of claim 6.

8. The expression vector of claim 7, wherein the expression vector is selected from the group consisting of a recombinant pET-28a (+) expression vector, a recombinant pET-30a (+) expression vector, and a recombinant pET-32a (+) expression vector.

9. A recombinant escherichia coli engineering bacterium, comprising the expression vector of claim 7 or 8, wherein the escherichia coli is selected from BL21 (DE 3).

10. The recombinant escherichia coli engineering bacterium according to claim 9, wherein the construction method of the recombinant escherichia coli engineering bacterium at least comprises the following steps:

s1, synthesizing the polynucleotide fragment according to claim 6;

s3, transforming the expression vector into escherichia coli BL21 (DE 3) to obtain the recombinant escherichia coli engineering bacteria.

11. A method for preparing a GLP-1/GIP receptor dual agonist derivative, comprising at least the steps of:

s4, culturing the recombinant escherichia coli engineering bacteria according to claim 9 or 10, and expressing the fusion protein;

s6, connecting a fatty acid side chain on the GLP-1/GIP receptor dual agonist polypeptide precursor to obtain a fatty acid modified derivative precursor;

the fatty acid side chain is HOOC- (CH) ₂ ) _14~20 -CO-, said side chain being attached to the epsilon amino group of lysine by a linker;

s7, modifying the N end of the fatty acid modified derivative precursor by a dipeptide group, wherein the dipeptide group is selected from His-Aib-or Tyr-Aib-; obtaining the GLP-1/GIP receptor dual agonist derivative.

12. The method of claim 11, wherein S6 comprises:

preparing a solution of the GLP-1/GIP receptor dual agonist polypeptide precursor and a fatty acid side chain precursor compound, respectively, and mixing the GLP-1/GIP receptor dual agonist polypeptide precursor and the fatty acid side chain precursor compound in a molar ratio of 1: 3-5, mixing;

preparing a solution of the GLP-1/GIP receptor dual agonist polypeptide precursor by taking water as a solvent, wherein the concentration is 4-6 mg/mL, and the pH is adjusted to 11.0-11.5; preparing a solution of the fatty acid side chain precursor compound with an organic solvent;

the organic solvent is acetonitrile.

13. The method of claim 11, wherein S7 comprises:

preparing a solution of a dipeptide group compound by adopting an organic solvent;

the dipeptide based compound is selected from Boc-His (Trt) -Aib-OH or Boc-Tyr (tBu) -Aib-OH; the organic solvent is acetonitrile;

combining the fatty acid modified derivative precursor with a dipeptide based compound in a molar ratio of 1: 2-4, terminating the reaction, separating, deprotecting and purifying to obtain the GLP-1/GIP receptor dual agonist derivative.

14. Use of a GLP-1/GIP receptor dual agonist derivative or a pharmaceutically acceptable salt thereof according to claim 1 or 2, or a pharmaceutical composition according to claim 3, for the preparation of a medicament for the treatment of metabolic diseases or weight loss.