CN114591416B - A kind of N-glycan modified glucagon-like peptide-1 analogue and its preparation method and application - Google Patents

Abstract

The invention discloses an N-glycan-modified glucagon-like peptide-1 analogue, a preparation method and application thereof. The present invention firstly discloses N-glycan-modified glucagon-like peptide-1 analogs, the amino acid sequence of which is shown in SEQ ID NO.1, and N-polysaccharide is modified on the asparagine residue of the amino acid sequence sugar. The present invention further discloses a preparation method of the glucagon-like peptide-1 analogue and its application in medicines for treating or preventing diabetes or obesity. The present invention carries out N-glycosylation modification on the glucagon-like peptide-1 analog according to a two-step enzymatic method, and adds a uniform sugar chain to the polypeptide chain of the GLP-1 analog to obtain N-glycan modified The GLP-1 analog has no change in the secondary structure compared with the natural GLP-1, has a longer half-life, enhanced proteolytic stability, and improved blood sugar-lowering effect, and has good application potential in the treatment or prevention of diabetes and obesity.

Description

A kind of N-glycan modified glucagon-like peptide-1 analogue and its preparation method and application

technical field

The invention relates to the technical field of medicine. More specifically, it relates to an N-glycan-modified glucagon-like peptide-1 analogue and its preparation method and application.

Background technique

Glucagon-like peptide-1 (GLP-1), a polypeptide hormone, is a promising therapeutic candidate in the treatment of type 2 diabetes. GLP-1 can produce physiological effects by binding to the GLP-1 receptor (G protein-coupled receptor). When the GLP-1 receptor is activated, it leads to a rapid increase in circulating adenosine monophosphate and intracellular calcium levels, resulting in Glucose-dependent insulin release, lowering blood sugar in the body. However, because it is easily degraded by enzymes in serum [mainly dipeptidyl peptidase Ⅳ (DPP-Ⅳ)], resulting in a short serum half-life (~2min), limiting its potential application in diabetes treatment (Ahrén, B. (2004) Enhancement or proliferation of GLP-1 activity as a strategy for treatment of type 2diabetes. Drug Discovery Today: Therapeutic Strategies 1, 207-212.).

Many attempts have been made to improve the pharmacological profile of GLP-1, including glycosylation, pegylation and lipidation (Moradi, S.V., Hussein, W.M., Varamini, P., Simerska, P., and Toth, I .(2016)Glycosylation,an effective synthetic strategy to improve the bioavailability of therapeutic peptides.Chem.Sci.7,2492-2500.;Frokjaer,S.,and Otzen,D.E.(2005)Protein drug stability:a formulation challenge.Nat Rev Drug Discov 4, 298-306.; Vaishya, R., Khurana, V., Patel, S., and Mitra, A.K. (2015) Long-term delivery of protein therapeutics. Expert Opin Drug Deliv 12, 415-440.).

Glycosylation modification is one of the most common protein post-translational modifications. Its method is mainly to combine a sugar chain with an amide in the asparagine (N-glycosylation) side chain of the polypeptide on the newly synthesized protein polypeptide connected. The glycosylation modification of protein drugs is a natural modification, which can profoundly affect the properties of proteins, such as protein folding, in vivo stability, immunogenicity and pharmacokinetics, etc. (Wu, Z.; Jiang, K.; Zhu, H.; Ma, C.; Yu, Z.; Li, L.; J.; Zhang, L.; Wang, P.G. (2016) Site-directed glycosylation of peptide/protein with homogeneous O-linked eukaryotic N-glycans. Bioconjug Chem. 27, 1972–1975.). The main feature of the glycosylated protein is the addition of sugar chains at specific positions on the original protein polypeptide, thereby improving the stability of the protein and reducing the degradation of the protein by proteases. On the other hand, the glycosylation modification of the polypeptide will increase the molecular weight of the original polypeptide drug, which can reduce the glomerular filtration rate. However, in the glycosylation modification, the oligosaccharide chain connected to the protein has a branched structure, and the anomeric carbon of each sugar group constituting the sugar chain has two possibilities: α and β, so the structure of the oligosaccharide chain is far more than that of the linear structure. Nucleic acid or polypeptide complex. The development of GLP-1 long-acting preparations is limited due to the large molecular weight, complex structure and lagging technology of uniform glycosylation modification of sugar chains.

Therefore, it is necessary to provide a new method that can add uniform sugar chains to the polypeptide chains of GLP-1 analogs, and obtain glycosylated GLP-1 analogs, so as to overcome the easy degradation of natural GLP-1 in vivo, The half-life is short and the hypoglycemic effect cannot be well exerted.

Contents of the invention

The first object of the present invention is to provide a kind of N-glycan modified glucagon-like peptide-1 (GLP-1) analog, to enhance the stability of GLP-1 analog, prolong its half-life, improve the The effect of blood sugar.

The second object of the present invention is to provide a method for preparing the above-mentioned glucagon-like peptide-1 analogue, so as to add uniform sugar chains to the polypeptide chain of the GLP-1 analogue.

The third object of the present invention is to provide a use of the above glucagon-like peptide-1 analog.

To achieve the above object, the present invention adopts the following technical solutions:

In a first aspect, the present invention provides an N-glycan modified glucagon-like peptide-1 (GLP-1) analog, the amino acid sequence of the glucagon-like peptide-1 analog is as shown in SEQ ID As shown in NO.1, and N-glycan is modified on the asparagine residue of the amino acid sequence.

Further, the N-glycans are complex N-glycans.

Further, the complex N-glycans are sialylated complex N-glycans (SCT) or desialylated complex N-glycans (CT).

Further, the N-glycan-modified glucagon-like peptide-1 analogue is a desialylated complex N-glycan-modified glucagon-like peptide-1 analogue ( Glycan-GLP-1 (G2) analog) and sialylated complex N-glycan modified glucagon-like peptide-1 analog (Glycan-GLP-1 (G2S2) analog) as shown in formula IV ):

The N-glycosylation modification site in the N-glycan modified glucagon-like peptide-1 analogue of the present invention is Asn ³⁴ , when Asn ³⁴ is replaced by Asn ²⁶ for glycosylation modification, the stability, activity and glucose stabilizing ability decreased, indicating that N-glycan modifications at different glycosylation sites have great differences in the stability, activity and glucose stabilizing ability of GLP-1 analogues, and in glucagon-like peptide The Asn ³⁴ position of the -1 analogue is glycosylated to modify the effect better.

The pharmaceutical composition comprising the above-mentioned N-glycan modified glucagon-like peptide-1 analog or its salt is also within the protection scope of the present invention.

Further, the pharmaceutical composition also includes pharmaceutically acceptable carriers and/or adjuvants.

In a second aspect, the present invention provides a method for preparing N-glycan-modified glucagon-like peptide-1 analogues, comprising modifying complex N-glycans to glucagon-like peptides using a two-step enzymatic method Peptide-1 analogs.

Further, the enzymes used in the two-step enzymatic method are transglycosylase and endonuclease mutant; preferably, the transglycosylase is ApNGT ^Q469A , and the endonuclease mutant is EndoCC ^N180H .

In a specific embodiment of the present invention, the preparation method of the N-glycan-modified glucagon-like peptide-1 analogue comprises the following steps:

Using ApNGT ^Q469A to modify the Glc residue to the glucagon-like peptide-1 analogue with the amino acid sequence shown in SEQ ID NO.1, to obtain the Glc-GLP-1 with the structure shown in Formula II:

Use EndoCC ^N180H to modify desialylated complex N-glycans and sialylated complex N-glycans to Glc-GLP-1, respectively, to obtain desialylated complex N-glycans modified with the structure of formula III Glucagon-like peptide-1 analogs and sialylated complex N-glycan-modified glucagon-like peptide-1 analogs with the structure shown in formula IV:

Further, the glucagon-like peptide-1 analogue is prepared by the ^following method: replaceable amino acid residues (i.e. ^{Lys 34} ) was replaced by Asn to introduce an N-glycosylation modification site (Asn ³⁴ ), Arg ³⁶ was replaced by Thr to obtain the sequence form of Asn-X-Thr, and the amino acid sequence was chemically synthesized to obtain glucagon as shown in SEQ ID NO.1 Peptide-1 analogs.

Further, the desialylated complex N-glycans (CT) and sialylated complex N-glycans (SCT) are prepared by the following method: use EndoM to digest the sialic acid glycopeptide with the structure shown in formula I to obtain Sialylated complex N-glycans (SCT); using BiNanH2 to digest sialylated complex N-glycans (SCT) to obtain desialylated complex N-glycans (CT);

Further, the use of ApNGT ^Q469A to modify Glc residues to GLP-1 analogues is accomplished by the following method: acceptor GLP-1 analogues and donor UDP-Glc at a molar ratio of 1:5 at pH 8.0 and a temperature of 37 Under the condition of ℃, react for 12h.

Further, the modification of desialylated complex N-glycans and sialylated complex N-glycans to Glc-GLP-1 using EndoCC ^N180H is accomplished by the following method: acceptor Glc-GLP-1 with a molar ratio of 1:10 GLP-1 reacted with the donor CT-oxa or SCT-oxa at pH 7.5 and 30°C for 15 minutes; further, the SCT-oxa was added to SCT by sequentially adding DMC, ddH ₂ O and Et ₃ N (SCT, The molar ratio of DMC and Et3N (1:15:45) was mixed and reacted on ice for 1 h. The preparation method of CT-oxa was the same as that of SCT-oxa.

In a third aspect, the present invention provides the application of the above-mentioned N-glycan modified glucagon-like peptide-1 analogue or pharmaceutical composition in the preparation of a medicament for treating or preventing diabetes and/or obesity.

The beneficial effects of the present invention are as follows:

The present invention carries out N-glycosylation modification (including sialylated complex N-glycan and desialylated complex N-glycan modification) on glucagon-like peptide-1 analogs according to a two-step enzymatic method. The enzymatic effect of glycosylase ApNGT ^Q469A and endonuclease mutant EndoCC ^N180H adds uniform sugar chains to the polypeptide chains of GLP-1 analogues to obtain sialylated complex N-glycan modified GLP-1 analogues GLP-1 analogs modified by desialylated complex N-glycans have no change in the secondary structure of natural GLP-1, prolong the half-life, enhance proteolytic stability, and improve the efficacy of lowering blood sugar. It has great application potential in diabetes and obesity.

Description of drawings

The specific implementation manners of the present invention will be further described in detail below in conjunction with the accompanying drawings.

Figure 1 shows the site-directed glycosylation modification of GLP-1 analogues and uniform complex N-glycans.

Figure 2 is the deep ultraviolet circular dichroism (CD) of natural GLP-1 and Glycan-GLP-1 analogs.

Figure 3 shows the biological activity of Glycan-GLP-1 analogues in type 2 diabetic db/db mice; the data are expressed as the mean ± standard deviation of 8 measurements.

Figure 4 shows the degree of hypoglycemia of Glycan-GLP-1 analogues relative to normal saline or natural GLP-1; wherein, a is the blood glucose level measured 30 minutes after the sugar administration, b is the blood glucose level measured 120 minutes after the sugar administration, and c is the glucose administration Blood glucose levels were measured 180 minutes later. Data are expressed as the mean ± standard deviation of 8 determinations. *P<0.05, **P<0.01, ***P<0.001, compared with normal saline. ^A P<0.05, ^B P<0.01, ^C P<0.001, compared with GLP-1. ^D P<0.05, compared with Glycan-GLP-1 (G2).

Figure 5 shows the biological activity of Glycan-GLP-1-2 analogues in type 2 diabetic db/db mice; the data are expressed as the mean ± standard deviation of 8 measurements.

Figure 6 shows the degree of hypoglycemia of Glycan-GLP-1-2 analogs relative to normal saline or natural GLP-1; wherein, a is the blood glucose level measured 30 minutes after the sugar administration, and b is the blood glucose level measured 120 minutes after the sugar administration. Data are expressed as the mean ± standard deviation of 8 determinations. *P<0.05, **P<0.01, ***P<0.001, compared with normal saline. ^A P<0.05, ^B P<0.01, ^C P<0.001, compared with GLP-1.

Detailed ways

In order to illustrate the present invention more clearly, the present invention will be further described below in conjunction with preferred embodiments and accompanying drawings. Similar parts in the figures are denoted by the same reference numerals. Those skilled in the art should understand that the content specifically described below is illustrative rather than restrictive, and should not limit the protection scope of the present invention.

Preparation of glucagon-like peptide-1 (GLP-1) analogs of embodiment 1N-glycan modification

1. Design glycosylation modification sites and synthesize GLP-1 analogs

Replace the replaceable amino acid residue (i.e. Lys ³⁴ ) on the C-terminus of Thr ¹³ to Val ³³ adjacent to the α-helix of the natural GLP-1 polypeptide chain with Asn to introduce the N-glycosylation modification site (Asn ³⁴ ), because The conserved Asn-X-Ser/Thr (X≠Pro) sequence required by ApNGT ^Q469A does not exist in natural GLP-1, so Arg ³⁶ is replaced by Thr to obtain the Asn-X-Thr sequence form, which is obtained by chemical synthesis GLP-1 analogs;

Wherein, the amino acid sequence of the natural GLP-1 is:

HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG (SEQ ID NO. 2)

The amino acid sequence of the GLP-1 analog is (wherein N is a glycosylation site):

HAEGTFTSDVSSYLEGQAAKEFIAWLVNGTG (SEQ ID NO. 1).

2. Preparation of complex N-glycans

Use EndoM and BiNanH2 to continuously digest sialoglycopeptides (SGP, whose structure is shown in formula I), purify and freeze-dry to obtain sialylated complex N-glycans (SCT) and desialylated complex N-glycans ( CT), and further prepare sialylated complex N-glycan oxazoline (SCT-oxa) and desialylated complex N-glycan oxazoline (CT-oxa), the specific steps are as follows:

Preparation of SCT by cleavage of SGP by EndoM: A reaction mixture containing 200 mM PBS (pH 6.5), 8 nM SGP and 10 μg EndoM was incubated overnight (12 h) at 30° C., and the reaction was terminated by boiling for 5 min. SCT was obtained by purifying and freeze-drying by high-performance liquid chromatography (HPLC) HILIC column.

Preparation of CT by cleavage of SCT by BiNanH2: A reaction mixture containing 200 mM Tris-HCl (pH 8.0), 8 nM SCT and 5 μg BiNanH2 was incubated overnight (12 h) at 37° C., and the reaction was terminated by boiling for 5 min. CT was obtained by purifying and freeze-drying by high-performance liquid chromatography (HPLC) HILIC chromatographic column.

Preparation of high-energy transition state SCT-oxa by SCT: take 5 μM SCT, add 45 μM DMC, 200 μL ddH ₂ O and 225 μM Et ₃ N (M _SCT : M _DMC : M _Et3N = 1:15:45) in sequence, mix and store on ice After reacting for 1 hour, SCT-oxa was obtained by Supelclean ^TM ENVI-Carb ^TM SPE Tube column purification and lyophilization (CT-oxa preparation method is the same as SCT-oxa);

3. Preparation of N-glycan-modified glucagon-like peptide-1 analogs (ie Glycan-GLP-1 analogs)

Complex N-glycans (CT and SCT) were modified into GLP-1 analogs by a two-step enzymatic method (transglycosylase ApNGT ^Q469A and endonuclease mutant EndoCC ^N180H ) respectively (as shown in Figure 1), Finally, Glycan-GLP-1 (G2) analogs and Glycan-GLP-1 (G2S2) analogs are obtained, and the specific steps are as follows:

Modification of Glc residues to GLP-1 analogues by ApNGT ^Q469A : A mixture containing 200 mM Tris-HCl (pH 8.0), 6 nM GLP-1 analogues (acceptor), 30 nM UDP-Glc (donor) and 20 μg ApNGT ^Q469A The reaction mixture was incubated overnight (12h) at 37°C and terminated by boiling for 5min. Carry out reverse-phase high-performance liquid chromatography (RP-HPLC) purification by C18 chromatographic column and analyze with ESI-MS, the result is as shown in Table 1, obtains intermediate product Glc-GLP-1, and structure is as shown in formula II:

Modification of complex N-glycans (CT, SCT) to GLP-1 analogs by EndoCC ^N180H : 200 mM PBS (pH 7.5), 6 nM Glc-GLP-1 analog as acceptor, 60 nM CT-oxa (or SCT-oxa) as a donor and 20 μg EndoCC ^N180H were mixed, incubated at 30° C. for 15 min, and the reaction was terminated by boiling for 5 min. Carry out reverse-phase high-performance liquid chromatography (RP-HPLC) purification by C18 chromatographic column, and purity is all greater than 95%, and is analyzed with electrospray ionization mass spectrometry (ESI-MS), and the result is as shown in table 1, has obtained desialylated Complex N-glycan-modified glucagon-like peptide-1 analogs (Glycan-GLP-1(G2) analogs) and sialylated complex N-glycan-modified glucagon-like peptide-1 are similar (Glycan-GLP-1 (G2S2) analog), its structure is shown in formula III and formula IV respectively:

Retention time, actual molecular weight and detection molecular weight of table 1 Glycan-GLP-1 analog

Among them, the specific steps of ESI-MS analysis are as follows: High-resolution mass spectrometry analysis uses Impact HD Q-ToF mass spectrometer (Bruker Daltonics), equipped with a heated ESI source and Ultra3000 high-performance liquid chromatography system (Thermo Fisher). HPLC purified samples can be detected by autosampler without separation. Full-scan positive mass spectrometry experiment (m/z range 300-2000; ESI voltage 4500V; nebulizer gas pressure 0.4bar; dry gas flow rate 4.0L/min, 180°C), using sodium formate as the internal mass calibration standard.

When the glycosylation modification by the above two-step enzymatic method is completed, each step is analyzed by RP-HPLC, and the conversion rate of the Glycan-GLP-1 analogue is calculated according to the peak area. The results are shown in Table 2. ApNGT ^Q469A can effectively transfer Glc residues to polypeptides, with a conversion rate of 94.44%; EndoCC ^N180H can modify the conversion rates of two complex N-glycans to Glc-GLP-1 respectively Both are above 60%, indicating that the total conversion rate of glycosylation modification by the two-step enzymatic method is higher than 50%, and the sialylation of N-glycans will not affect the conversion rate of glycosylation modification, which can effectively complete the GLP- 1 analog site-directed glycosylation modification.

The conversion rate of the Glycan-GLP-1 analogue synthesized by Table 2ApNG ^TQ469A and EndoCC ^N180H

Example 2 Circular Dichroism Analysis of the Secondary Structure of Glycan-GLP-1 Analogs

The secondary structure (195-260nm) of Glycan-GLP-1 analogues was determined in the range of far-ultraviolet (195-260nm) by using far-ultraviolet circular dichroism (CD), and compared with natural GLP-1, the specific steps are as follows : Spectra of 120 μM GLP-1 or Glycan-GLP-1 analogs were recorded on a J-810 Spectropolarimeter (Jasco) at ambient temperature (25° C.) using 0.1 cm path length quartz cuvettes. All samples were scanned at least 3 times with the background signal from the buffer.

The results are shown in Figure 2, and the results show that the CD spectrum of Glycan-GLP-1 (G2) and Glycan-GLP-1 (G2S2) is not significantly different from that of natural GLP-1, indicating that N-glycan modification has a positive effect on the effect of GLP-1. Secondary structure has no effect.

Example 3 The effect of DPP-IV on the proteolytic stability of Glycan-GLP-1 analogs In vitro, DPP-IV was used to evaluate the proteolytic stability of GLP-1 and two Glycan-GLP-1 analogs, specific steps As follows: DPP-IV was diluted with 50 mM Tris-HCl (pH 7.4) to a final concentration of 10 ng/μL. In order to ensure the accuracy of the half-life measurement, first optimize the amount of DPP-IV required so that the in vitro half-life of GLP-1 is about 10 minutes. To this end, 0.3 mol (ie 1 μg) of native GLP-1 was incubated with different doses of DPP-IV at 37°C to determine the appropriate DPP-IV dose. Then, an equal amount of DPP-IV was incubated with every 0.3 mol of Glycan-GLP-1 analog, and its in vitro half-life was determined. Each sample was tested in triplicate. All incubation mixtures were boiled for 5 min to terminate proteolysis, and the supernatant was collected after centrifugation at 12000 rpm for 30 min, and analyzed by RP-HPLC. The proteolytic half-life of each Glycan-GLP-1 analog was calculated based on the peak area.

The results are shown in Table 3. The results showed that the in vitro half-lives of Glycan-GLP-1(G2) analogs and Glycan-GLP-1(G2S2) analogs were both longer than those of natural GLP-1, but to different degrees. The in vitro half-lives of Glycan-GLP-1(G2) analogs and Glycan-GLP-1(G2S2) analogs are 17.8 times and 24.0 times that of natural GLP-1, respectively, which significantly enhance their proteolytic stability and overcome natural GLP -1 for the short half-life issue. In addition, sialylation contributes greatly to proteolytic stability, and the half-life of Glycan-GLP-1(G2S2) analogues is 1.3 times longer than that of Glycan-GLP-1(G2) analogues, which is attributed to the negative The charge prevented the peptide from binding to DPP-IV, indicating that N-glycan modifications, especially sialylated complex N-glycan modifications, have great potential in improving the proteolytic stability of GLP-1.

Table 3 GLP-1 and Glycan-GLP-1 analogues incubated with DPP-IV in vitro half-life

Example 4 Type 2 Diabetic db/db Mice Evaluate the Glucose Stabilization Ability of Glycan-GLP-1 Analogues

The glucose stabilizing ability of Glycan-GLP-1 analogs was evaluated by performing an oral glucose tolerance test (OGTT) on type 2 diabetic db/db mice, and compared with natural GLP-1 and normal saline, the specific steps are as follows:

传感器，美国罗氏诊断公司)测量血糖水平。最终通过(i)最高血糖水平(BGL_max)、(ii)血糖降至10mmol/L以下所需时间(T_BGL<10mmol/L)和(iii)总低血糖程度(HGD％_Total)三个因素来评价血糖稳定能力。总低血糖程度(与生理盐水组比较％)计算如下：[(AUC_{saline,0–180min}-AUC_{test,0–180min})/AUC_{saline,0–180min}]×100(AUC：曲线下面积)。After fasting for 18 hours, BKS-Lepr ^em2Cd479 /Jpt db/db mice (7-10 weeks old) were intraperitoneally injected with normal saline, GLP-1 or Glycan-GLP-1 analogs (10nmol /kg, n=8). At 0 min, each mouse was given 1.0 g/kg glucose. At predetermined times (15min, 30min, 60min, 90min, 120min and 180min), a drop of blood was drawn from the tail vein, and a one-touch blood glucose meter (

Sensor, Roche Diagnostics, USA) measures blood glucose levels. Three factors are finally passed (i) the highest blood glucose level (BGL _max ), (ii) the time required for blood glucose to drop below 10mmol/L (T _BGL<10mmol/L ) and (iii) the total hypoglycemia degree (HGD% _Total ) To evaluate blood sugar stability. The total hypoglycemia degree (% compared with the normal saline group) was calculated as follows: [(AUC _{saline,0–180min} -AUC _{test,0–180min} )/AUC _{saline,0–180min} ]×100 (AUC: area under the curve).

The results of the blood sugar stabilizing ability of Glycan-GLP-1 analogs are shown in Table 4, and the biological activity in type 2 diabetic db/db mice is shown in Figure 3, relative to the degree of hypoglycemia of normal saline or natural GLP-1 As shown in Fig. 4, the results show that both Glycan-GLP-1 analogs have significant glucose stabilizing ability. Glycan-GLP-1 analogs have more rapid glucose stabilization ability, and the BGL _max of Glycan-GLP-1(G2) analogs and Glycan-GLP-1(G2S2) analogs were 14.50±1.62mmol/L and 15.50± 3.74mmol/L, significantly lower than normal saline (29.73±2.56mmol/L) and GLP-1 (19.27±1.41mmol/L). Glycan-GLP-1(G2) analogues and Glycan-GLP-1(G2S2) analogues lowered blood glucose faster than normal saline (P<0.001) and natural GLP-1 (P<0.001 and P<0.001, respectively) within 30 min. <0.01) (shown in a in Figure 3 and Figure 4), and can reduce blood sugar to <10mmol/L within 60min after sugar administration, while _{TBGL<10mmol/L} of normal saline and natural GLP-1 is 180min (shown in Table 4 and Figure 3). In addition, the glucose stabilizing ability of Glycan-GLP-1 analogues has persistence, 120min (Glycan-GLP-1(G2) analogues and Glycan-GLP-1(G2S2) analogues) and 180min (Glycan-GLP-1(Glycan-GLP-1( G2S2) analogs) were significantly more effective than native GLP-1 (shown in b and c in Figure 4). The above results show that N-glycan modification can effectively improve the therapeutic effect of polypeptide drugs, and has a good application prospect in the treatment of type 2 diabetes.

Table 4 Glucose stabilization ability in vivo of GLP-1 and Glycan-GLP-1 analogs

^a BGL _max : maximum blood glucose level; ^b T _BGL<10mmol/L : time required for blood glucose to drop below 10mmol/L; ^c HGD% _total : total hypoglycemia degree; ^dNA : not applicable.

In addition, the sialylation of N-glycans also has a considerable impact on the biological activity of peptides, and the glucose stabilization effect of Glycan-GLP-1(G2S2) analogues at 120min and 180min was significantly better than that of Glycan-GLP-1(G2) Analogs (shown in b and c in Figure 4). Due to the longer half-life of Glycan-GLP-1 (G2S2) analogues, it has better blood sugar stabilization ability.

Comparative example 1

Replace the N-glycosylation modification site (Asn ³⁴ ) in Example 1 with Asn ²⁶ for glycosylation modification, the method is the same as in Example 1, and the desialylated complex N-glycan modified glucagon obtained Glucagon-like peptide-1 analogs (Glycan-GLP-1-2 (G2) analogs) and sialylated complex N-glycan modified glucagon-like peptide-1 analogs (Glycan-GLP-1-2 (G2S2) analogs).

Utilize the method described in Example 3 to detect the proteolytic stability of DPP-IV to Glycan-GLP-1-2 (G2) analogs and Glycan-GLP-1-2 (G2S2) analogs and use the method described in Example 4 to detect Glucose stabilization ability of Glycan-GLP-1-2 (G2) analogs and Glycan-GLP-1-2 (G2S2) analogs in type 2 diabetes db/db mice, the results are shown in Table 5, 6 and Figure 5 , 6, the results show that: when the glycosylation modification site is replaced by Asn ²⁶ , the stability of Glycan-GLP-1-2 (G2S2) analogs is significantly increased, and the half-life is 374.7min, which is natural GLP-1 ( 10.2min) 36.7 times; however, compared with natural GLP-1, the biological activity has decreased, indicating that the modification of N-glycans at this site will affect the biological activity of GLP-1 analogs, while Glycan-GLP- 1-2(G2S2) analogues have lower activity than Glycan-GLP-1-2(G2) analogues, that is, sialylation will further weaken the activity, and their BGL _max are higher than natural GLP-1, respectively 25.71 ±3.19mmol/L and 24.07±3.79mmol/L, in addition, _{TBGL<10mmol/L} is only equivalent to natural GLP-1, far less than the glucose stabilizing effect after glycosylation modification at the Asn ³⁴ site.

Therefore, N-glycan modifications at different glycosylation sites have great differences in the stability, activity and glucose stability of GLP-1 analogs, and the effect of glycosylation modification at Asn ³⁴ is better .

Table 5 In vitro half-life of Glycan-GLP-1-2 analogs incubated with DPP-IV

Table 6 Glycan-GLP-1-2 analogues glucose stabilizing ability in vivo

^a BGL _max : maximum blood glucose level; ^b T _BGL<10mmol/L : time required for blood glucose to drop below 10mmol/L; ^c HGD% _total : total hypoglycemia degree; ^dNA : not applicable.

In summary, the GLP-1 analogs provided by the present invention with N-glycan modification at the Asn ³⁴ position, especially the GLP-1 analogs with sialylated complex N-glycan modification, can effectively resist DPP- IV degrades, prolongs half-life, improves glucose stability, and has very good application potential.

Apparently, the above-mentioned embodiments of the present invention are only examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. Those of ordinary skill in the art can also make It is impossible to exhaustively list all the implementation modes here, and any obvious changes or changes derived from the technical solutions of the present invention are still within the scope of protection of the present invention.

SEQUENCE LISTING

<110> Hebei University of Science and Technology

<120> JLP22I0144

<130> An N-glycan-modified glucagon-like peptide-1 analogue and its preparation method and application

<160> 2

<170> PatentIn version 3.5

<210> 1

<211> 31

<212> PRT

<213> Artificial Sequence

<400> 1

His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly

1 5 10 15

Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Asn Gly Thr Gly

20 25 30

<210> 2

<211> 31

<212> PRT

<213> Artificial Sequence

<400> 2

His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly

1 5 10 15

Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly

20 25 30

Claims (5)

1. An N-glycan modified glucagon-like peptide-1 analogue is characterized in that the amino acid sequence of the glucagon-like peptide-1 analogue is shown as SEQ ID NO.1, and N-glycan is modified on asparagine residue of the amino acid sequence, and the specific structure is a desialylated complex N-glycan modified glucagon-like peptide-1 analogue with the structure shown as formula III or a sialylated complex N-glycan modified glucagon-like peptide-1 analogue with the structure shown as formula IV:

2. a pharmaceutical composition comprising the glucagon-like peptide-1 analog of claim 1, or a salt thereof.

3. The pharmaceutical composition of claim 2, further comprising a pharmaceutically acceptable carrier and/or adjuvant.

4. The method of claim 1, comprising modifying an N-glycan into the glucagon-like peptide-1 analog using a two-step enzymatic process;

the method specifically comprises the following steps:

using ApNGT ^Q469A Modifying Glc residue to glucagon-like peptide-1 analogue with amino acid sequence shown as SEQ ID NO.1 to obtain Glc-GLP-1 with structure shown as formula II:

using Endocc ^N180H Respectively modifying the desialylated complex N-glycan and the sialylated complex N-glycan to Glc-GLP-1 to respectively obtain a desialylated complex N-glycan modified glucagon-like peptide-1 analogue with a structure shown as a formula III and a sialylated complex N-glycan modified glucagon-like peptide-1 analogue with a structure shown as a formula IV:

5. use of a glucagon-like peptide-1 analog of claim 1 or a pharmaceutical composition of claim 2 or 3 in the manufacture of a medicament for the treatment or prevention of diabetes and/or obesity.

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