A polypeptide analogue having GLP-1 receptor agonistic and GIP receptor antagonistic activity and uses thereof

Abstract

This invention discloses a polypeptide analog with GLP-1 receptor agonist and GIP receptor antagonist activities and its applications. The sequence structure of the polypeptide analog is selected from any of the amino acid sequences shown in SEQ ID NO:1-2. This polypeptide compound achieves excellent hypoglycemic and sustained, superior weight loss effects by agonizing the GLP-1 receptor and antagonizing the GIP receptor. Therefore, this type of polypeptide compound has broad application prospects in the field of drug preparation for the prevention and treatment of metabolic syndromes, such as diabetes and obesity.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a polypeptide analog with GLP-1 receptor agonist and GIP receptor antagonist activities and its applications. Background Technology

[0002] With the continued rise in the incidence of obesity and related metabolic diseases, type 2 diabetes mellitus (T2DM) has become a major threat to global public health. Obesity is one of the most significant risk factors for T2DM. While current mainstream drugs, glucagon-like peptide-1 (GLP-1) receptor agonists, have made progress in lowering blood sugar and reducing weight, limitations remain in terms of the sustainability of efficacy and control of side effects. On the one hand, the weight loss effect of GLP-1 receptor agonists has an upper limit, and significant weight rebound is common after discontinuation. On the other hand, high-dose use can also cause significant gastrointestinal adverse reactions, affecting long-term adherence. Therefore, there is an urgent need to develop new drugs that offer better weight and blood sugar management with fewer side effects.

[0003] In recent years, multi-target peptide drugs have become an important direction in this field. Besides the GLP-1 receptor, the incretin glucose-dependent insulinotropic peptide (GIP) receptor has also been shown to play an important role in energy metabolism and weight regulation. However, recent studies have found that in addition to activating GIP receptors for weight control, GIP receptor antagonists can also effectively combat obesity induced by a high-fat diet, and in animal models, when used in combination with GLP-1 receptor agonists, they exhibit a synergistic effect in inhibiting weight gain and providing sustained metabolic improvement. Some studies suggest that chronic GIP receptor activation may even lead to receptor desensitization and functional antagonism, while direct antagonism of GIP receptors holds promise for achieving better and more sustained weight loss, and reducing GLP-1 receptor agonist-related side effects and rebound weight gain after discontinuation.

[0004] However, current research on bifunctional peptides with both GLP-1 receptor agonist and GIP receptor antagonist activities is limited, and peptide molecules capable of simultaneously achieving highly efficient GLP-1 receptor agonist activity and excellent GIP receptor antagonist activity are very limited. The challenge in peptide design and modification lies in ensuring that the agonist effect on the GLP-1 receptor remains unaffected while effectively blocking GIP receptor signaling. Therefore, novel bifunctional peptides represent a key innovative direction in the current development of drugs for obesity and type 2 diabetes mellitus (T2DM). In summary, developing peptide analogs with dual GLP-1 receptor agonist and GIP receptor antagonist effects could contribute to achieving better glycemic control and sustained weight loss, providing new solutions for the treatment of obesity and T2DM. Summary of the Invention

[0005] This invention provides a peptide analog with GLP-1 receptor agonist and GIP receptor antagonist activities and its applications. By simultaneously agonizing GLP-1 receptors and antagonizing GIP receptors, this peptide analog can achieve excellent hypoglycemic and sustained, superior weight loss effects. This peptide analog shows a broader application prospect in the field of preparing drugs for the prevention and treatment of metabolic syndromes, such as diabetes and obesity.

[0006] To achieve the above-mentioned objectives, the technical solution of the present invention is as follows: A polypeptide analog having GLP-1 receptor agonist and GIP receptor antagonist activities, wherein the sequence structure of the polypeptide analog is selected from any of the amino acid sequences shown in SEQ ID NO:1-2: SEQ ID NO: 1 SEQ ID NO: 2 .

[0007] The present invention also provides a pharmaceutical composition prepared from a polypeptide analog having GLP-1 receptor agonist and GIP receptor antagonist activities, the pharmaceutical composition comprising the aforementioned polypeptide analog having GLP-1 receptor agonist and GIP receptor antagonist activities and a pharmaceutically acceptable carrier, diluent or excipient.

[0008] The present invention also provides the use of the above-mentioned polypeptide analog or the above-mentioned pharmaceutical composition in the preparation of a medicament for treating metabolic diseases or conditions, said metabolic diseases or conditions being obesity and diabetes.

[0009] Compared with the prior art, the beneficial technical effects of the present invention are as follows: GLP-1 receptor agonists generally suffer from insufficient weight loss and weight rebound after discontinuation. The peptide analogs of this invention, which have GLP-1 receptor agonist and GLP-1 receptor antagonist activities, not only have excellent hypoglycemic and weight loss activities, but also maintain a long-term weight loss effect after discontinuation. This largely overcomes the defect of rapid weight rebound after discontinuation of GLP-1 drugs and solves the pain point of clinical application of such drugs. Attached Figure Description

[0010] Figure 1 This is the blood glucose-time curve of the oral glucose tolerance test of the polypeptide analog of the present invention in Kunming mice.

[0011] Figure 2 The percentage change in body weight of the polypeptide analog of the present invention after 49 days of long-term administration to DIO mice. Detailed Implementation

[0012] The present invention will now be described in detail with reference to the accompanying drawings and embodiments.

[0013] Unless otherwise defined in this invention, the scientific and technical terms used in this specification shall have the meanings commonly understood by one of ordinary skill in the art.

[0014] Furthermore, the amino acid sequence of the polypeptide analog described in this invention includes the standard amino acid codes (including three-letter and single-letter codes) commonly used in this invention, as well as the non-natural amino acid α-aminoisobutyric acid (Aib). The structure of the non-natural amino acid is described below: Furthermore, unless otherwise specified, all amino acid residues in the polypeptides of the present invention are preferably in the L configuration.

[0015] Furthermore, the "-NH2" portion at the C-terminus of the sequence indicates an amide group (-CONH2) at the C-terminus.

[0016] To illustrate the present invention in more detail, the following specific embodiments are provided in this specification, but the embodiments of the present invention are not limited thereto. Example 1

[0017] Synthesis of SEQ ID NO: 1 The target peptide compound of this invention was prepared using the Fmoc solid-phase peptide synthesis method. Using Rink Amide MBHA resin as the solid-phase support, Fmoc-Lys(Dde)-OH was first coupled to the resin as the starting amino acid for synthesis. After coupling, the Fmoc protecting group was removed using a 20% piperidine / DMF system to obtain the free amino group. Subsequently, the amino acid residues required for the GIP receptor antagonistic sequence (lower structural sequence) were added sequentially. Each coupling step used an HBTU / HOBt / DIPEA condensation system, with a reaction time of 1-2 hours. For the Lys residues required for fatty acid modification, Fmoc-Lys(Mtt)-OH was used for introduction. After the GIP receptor antagonistic sequence was synthesized, the Dde protecting group of the first C-terminal Lys residue was removed using 2% hydrazine hydrate / DMF. After Dde removal, the side chain of this Lys residue... The GLP-1 receptor agonist sequence (upper structural sequence) was synthesized stepwise from the amino group, using the same coupling and deprotection methods as described above. After the complete sequence synthesis, the Mtt protecting group on the Lys residue was removed using 1% trifluoroacetic acid. Subsequently, side-chain fatty acid modification was performed sequentially, introducing Fmoc-OEG-OH, Fmoc-OEG-OH, Fmoc-Glu(OtBu)-OH, and tert-butyl octadecanoate modifying groups. After all reactions were completed, the synthesized peptide was completely deprotected and cleaved, and the crude product was collected. The crude peptide sample was purified by semi-preparative HPLC and then lyophilized to obtain the purified target peptide. The molecular weight of the obtained peptide was determined by ESI-MS, and the theoretical molecular weight was 9105.15. ESI-MS m / z: calculated value [M+7H] 7+ 1301.7, [M+8H] 8+ 1139.1; Observation [M+7H] 7+ 1301.3, [M+8H] 8+ 1139.0. Example 2

[0018] Synthesis of SEQ ID NO: 2 The synthesis method was the same as in Example 1. The molecular weight of the target peptide analog was confirmed by mass spectrometry, with a theoretical relative molecular mass of 9105.15. ESI-MS m / z: Calculated value [M+7H] 7+ 1301.7, [M+8H] 8+ 1139.1; Observation [M+7H] 7+ 1301.5, [M+8H] 8+ 1138.9. Example 3

[0019] GLP-1 receptor agonist and GIP receptor antagonist activities of peptide analogs HEK293 cell lines stably expressing the GLP-1 receptor were seeded in DMEM medium supplemented with 10% fetal bovine serum (100 μg / mL Hygromycin B) and cultured routinely at 37°C in a 5% CO2 incubator. After cells reached 80% confluence, they were digested with Cell Dissociation Buffer, collected, and counted. Cell suspension was aliquoted into 384-well plates at a density of 9 μL per well. The peptide compounds to be used were first dissolved in DMSO and then serially diluted 10-fold with Stimulation Buffer to prepare working solutions. 1 μL of each gradient peptide compound solution was added to each well, mixed, and centrifuged to ensure adequate drug contact with the cells. The solutions were then incubated at 37°C for 30 minutes. Subsequently, Eu-cAMP and Ultra-anti-cAMP were diluted to working concentrations with Detection Buffer, and 5 μL of each was added to the corresponding wells. The solutions were mixed, centrifuged again, and incubated at room temperature for 1 hour to complete the reaction. After the reaction, fluorescence signal values ​​at wavelengths of 665 nm and 620 nm were acquired using a Perkin Elmer multi-mode microplate reader. Using the 665 / 620 nm value as the response signal, a curve was plotted showing the relationship between peptide concentration and the ratio (665 / 620 nm). Nonlinear regression analysis was performed using GraphPad Prism software to calculate and obtain the EC50 of the peptide compound. 50 Numerical value.

[0020] The culture method for the HEK293 cell line stably expressing the GIP receptor and the dilution method for the peptide compound were the same as those for the GLP-1 receptor detection procedure. The specific experimental procedures were as follows: HEK293 cells cultured to 80% confluence and stably expressing the GIP receptor were digested, counted, and aliquoted into 384-well plates at 9 μL per well. The peptide compound was first dissolved in DMSO, then serially diluted 10-fold with Stimulation Buffer to prepare working solutions of various concentrations. 1 μL of the peptide compound sample solution was added to each well, mixed, centrifuged, and incubated at 37°C for 30 minutes. Then, 50 pM of human GIP was added to each well, and the cells were incubated at 37°C for another 30 minutes to allow the GIP to fully act on the cells. Next, Eu-cAMP and Ultra-anti-cAMP were diluted to working concentrations with Detection Buffer, and 5 μL of each were added to the corresponding wells, mixed, centrifuged, and incubated at room temperature for 1 hour to complete the detection reaction. Finally, the fluorescence signals at 665 nm and 620 nm wavelengths in each well were detected using a Perkin Elmer multi-plate reader. The reaction effect was characterized by the fluorescence ratio (665 / 620 nm), and a curve showing the relationship between peptide concentration and the ratio was plotted. Using GraphPad Prism software, the IC50 of the peptide compound's antagonistic activity against the GIP receptor was calculated and obtained by fitting the curve using a nonlinear regression method. 50 value.

[0021] Table 1: GLP-1 receptor agonists (ECG) of the peptide analogs of the present invention 50 ) and GIP receptor antagonistic activity (IC) 50 ) As shown in Table 1, both SEQ ID NO: 1 and SEQ ID NO: 2 of the present invention exhibit strong GLP-1 receptor agonist activity, especially SEQ ID NO: 2, whose GLP-1 receptor agonist activity is superior to that of GLP-1. Furthermore, the GIP receptor antagonist activity demonstrates that SEQ ID NO: 1 and SEQ ID NO: 2 of the present invention also possess excellent GIP receptor antagonist activity, particularly SEQ ID NO: 2, whose GIP receptor antagonist activity is superior to that of the GIP receptor antagonist GIP(3-30)NH2. Example 4

[0022] Acute hypoglycemic activity of peptide analogs Male Kunming mice (weighing 22–28 g) were acclimatized for one week. One day before the experiment, all mice were randomly divided into three groups of six. Mice in each group were fasted for 12 hours prior to the experiment but allowed free access to water. On the day of the experiment, tail blood was collected from each group 60 minutes before the glucose load to measure basal blood glucose levels. Subsequently, mice were subcutaneously injected with either saline or semaglutide at a dose of 30 nmol / kg, and SEQ ID NO: 2. Sixty minutes later (recorded as 0 minutes), all mice were orally administered a 3 g / kg glucose solution. Tail blood was collected at different time points after the glucose load, and the blood glucose concentration was measured immediately using a blood glucose meter. Blood glucose-time curves were plotted based on the portable blood glucose meter data at different time points for each group.

[0023] like Figure 1 As shown, SEQ ID NO: 2 of the present invention has excellent acute hypoglycemic activity, and its hypoglycemic effect is superior to that of the marketed GLP-1 drug semaglutide. Example 5

[0024] Long-term weight loss and sustained weight loss after discontinuation of peptide analogs The DIO mouse model was established as follows: Six-week-old male C57bL / 6 mice were fed a high-fat diet (ResearchDiets, D12492, 60% fat content) for approximately 18 weeks. Mice weighing over 45 g were selected for the treatment group. DIO mice were divided into groups of six based on weight and housed individually. Each group of mice was subcutaneously administered saline (blank group), 30 nmol / kg semaglutide, and SEQ ID NO: 2 every two days for 21 days. After treatment, the mice were discharged and kept in the rearing group for another 49 days to assess weight rebound. The weight of each group of mice was measured at fixed times daily.

[0025] like Figure 2 As shown, SEQ ID NO: 2 of the present invention exhibits significantly better weight loss than semaglutide during a 21-day dosing cycle. More importantly, within a 28-day period after discontinuation of dosing, the weight of semaglutide mice rebounded rapidly, becoming nearly indistinguishable from the control group by day 49. However, the weight of mice in the SEQ ID NO: 2 group did not rebound significantly within the 28 days after discontinuation of dosing (i.e., days 22-49 of the experimental cycle), demonstrating a significantly more sustained weight loss effect than semaglutide. This indicates that SEQ ID NO: 2 not only has excellent weight loss effects but also maintains a very long-term weight maintenance effect after discontinuation, showing potential to overcome the rapid rebound defect of GLP-1 drugs after discontinuation.

Claims

1. A polypeptide analogue having GLP-1 receptor agonist and GIP receptor antagonist activity, characterised in that, the sequence structure of the polypeptide analog is selected from any one of the amino acid sequences as shown in SEQ ID NO: 1-2: SEQ ID NO: 1 SEQ ID NO: 2 。 2. A pharmaceutical composition prepared from a polypeptide analogue having GLP-1 receptor agonistic and GIP receptor antagonistic activity, characterized in that, The pharmaceutical composition comprises the polypeptide analog having GLP-1 receptor agonizing and GIP receptor antagonizing activities as claimed in claim 1 and a pharmaceutically acceptable carrier, diluent or excipient.

3. Use of the polypeptide analog as claimed in claim 1 or the pharmaceutical composition as claimed in claim 2 in the preparation of a medicament for treating metabolic diseases or disorders.

4. Use according to claim 3, characterized in that, The metabolic diseases or disorders are obesity and diabetes.

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