A polypeptide having GLP-1 receptor and GIP receptor biased agonism and uses thereof

Abstract

The application discloses a polypeptide with GLP-1 receptor and GIP receptor biased agonism and application thereof. The polypeptide has high agonistic activity to GLP-1 receptor and GIP receptor and a significant signal biased agonistic effect, and can synergistically enhance the biological functions of GLP-1 receptor and GIP receptor. The polypeptide not only has a GLP-1 receptor-mediated hypoglycemic effect, but also has the effects of GIP receptor regulation of sugar and fat metabolism and inhibition of appetite, and shows more excellent curative effects in terms of hypoglycemia, blood lipid regulation and weight loss, and has lower gastrointestinal side effects. Therefore, the polypeptide has wide application prospects and great potential in preparation of drugs for treating metabolic syndrome related diseases (such as diabetes, obesity and dyslipidemia).

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a polypeptide with biased agonistic activity against GLP-1 and GIP receptors and its uses. Background Technology

[0002] Currently, the global incidence of obesity is rising, and at the same time, the prevalence of type 2 diabetes mellitus (T2DM) is also increasing significantly. To address these health issues, the biopharmaceutical and medical communities are continuously dedicated to developing therapeutic drugs that can simultaneously lower blood sugar and weight. Since the U.S. Food and Drug Administration (FDA) approved exenatide for glycemic control in patients with T2DM in 2005, glucagon-like peptide-1 (GLP-1) receptor agonists, such as liraglutide and semaglutide, have been successively approved for marketing and widely used in clinical practice. These GLP-1 receptor (GLP-1R) agonists have greatly improved glycemic management in patients with T2DM. Furthermore, liraglutide and semaglutide, at higher doses, were approved for weight management in 2014 and 2021, respectively, further solidifying the important role of GLP-1R agonists in the treatment of T2DM and obesity.

[0003] Existing research indicates that after eating, GLP-1 and glucose-dependent insulinotropic peptide (GIP) are secreted by L cells and K cells in the gut, respectively, and work synergistically to regulate postprandial blood glucose levels, energy intake, and metabolic function. Based on this, researchers developed tirzepatide, a drug that simultaneously activates the GIP receptor (GIPR) and GLP-1R, classifying it as a dual-target receptor agonist. In clinical trials, tirzepatide demonstrated significant blood glucose-lowering and weight-loss effects in patients with type 2 diabetes mellitus (T2DM), and its weight-loss effect in obese patients was superior to that of single GLP-1R receptor agonists (such as semaglutide). Existing literature reports that while tirzepatide can mimic the role of endogenous GIP on GIPR in in vitro experiments, it exhibits different characteristics from its natural ligand in the GLP-1R-mediated signal transduction mechanism: it is more inclined to induce the production of cyclic adenosine monophosphate (cAMP) and less recruits β-arrestin, exhibiting a biased agonism towards GLP-1R signaling. This biased signal transduction may promote the retention of GLP-1R on the cell membrane surface, thereby enhancing the clinical efficacy of the drug (JCI Insight, 2020, 5, e140532). Numerous studies have confirmed that molecules with biased signaling agonists of GLP-1R are superior to non-biased agonists in lowering blood glucose.

[0004] In the field of GLP-1R agonists, studies have shown that reducing β-arrestin recruitment helps improve glucose-lowering and weight-loss effects. Meanwhile, research has confirmed that biased activation of GIPR also plays a crucial role in maintaining glycemic stability, regulating food intake, and weight management. Similar to GLP-1R, GIPR is also involved in energy intake and weight regulation in the central nervous system. By synergistically and biasedly activating GLP-1R and GIPR, superior therapeutic effects can be achieved. In conclusion, biased activation of GLP-1R not only improves therapeutic outcomes, but biased activation of GIPR is equally important. Dual-biased activation of GLP-1R and GIPR holds promise for providing a superior treatment option for T2DM and obesity (Cell Reports Medicine, 2025, 6, 102156). Summary of the Invention

[0005] The purpose of this invention is to provide a polypeptide with biased agonist activity towards GLP-1 and GIP receptors and its uses. This polypeptide can simultaneously stimulate both GLP-1 and GIP receptors, exhibiting biased signaling agonist activity towards these two receptors (i.e., more inclined to induce cAMP production and less recruiting β-arrestin). This allows for the maximization of the biological activities of GLP-1 and GIP, possessing not only the therapeutic effects of GLP-1 on diabetes but also the beneficial effects of GIP on glucose and lipid metabolism and appetite suppression. Furthermore, it exhibits superior hypoglycemic, lipid-regulating, and weight-loss effects with fewer gastrointestinal side effects, making it a promising candidate for the preparation of drugs for treating metabolic syndromes such as diabetes, obesity, and dyslipidemia.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a polypeptide or a pharmaceutically acceptable salt thereof having biased agonistic activity against GLP-1 and GIP receptors, wherein the amino acid sequence of the polypeptide is of the following general formula: His-Xaa1-Glu-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Ile-Xaa2-Leu-Asp-Lys-Ile-Ala-Gln-Lys-Al a-Phe-Val-Gln-Trp-Leu-Ile-Ala-Gly-Gly-Pro-Pro-Xaa3-Arg-Ala-Ile-Val-Val-Ser-Xaa4-NH2; in, Xaa1 is derived from Ala or Aib; Xaa2 is derived from Ile or Aib; Xaa3 is derived from Pro or Aib; Xaa4 is derived from Lys or Lys with modified side chains; The structural formula of Lys with modified side chains is shown below: .

[0007] In some possible implementations, the amino acid sequence of the polypeptide is as follows: SEQ ID NO: 1 .

[0008] In a second aspect, the present invention also provides a pharmaceutical composition, characterized in that the pharmaceutical composition comprises the polypeptide described in any one of the first aspects and a pharmaceutically acceptable carrier, diluent or excipient.

[0009] Thirdly, the present invention also provides the use of the polypeptide having GLP-1 receptor and GIP receptor biased agonist activity as described in the first aspect, or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition described in the second aspect, in the preparation of a medicament for treating metabolic diseases or conditions.

[0010] In some possible implementations, the metabolic disease or condition includes diabetes, obesity, and dyslipidemia.

[0011] Compared with the prior art, the beneficial technical effects of the present invention are as follows: (1) The polypeptide compound provided by this invention possesses excellent GLP-1 receptor and GIP receptor agonist activity. Its agonist efficacy against GLP-1 receptor is approximately 12 times higher than that of tirzepatide, the best existing drug in its class, and its agonist efficacy against GIP receptor is approximately 4 times higher than that of tirzepatide. Furthermore, the polypeptide compound of this invention also exhibits biased signaling agonist activity against both GLP-1 and GIP receptors, i.e., it is more inclined to induce cAMP production while reducing the recruitment of β-arrestin. Compared with tirzepatide, which only exhibits biased signaling agonist activity against GLP-1 receptor, the polypeptide compound of this invention shows superior effects in weight loss, blood sugar reduction, and lipid regulation, and significantly reduces the occurrence of gastrointestinal side effects. (2) The polypeptide compound of the present invention, by introducing the PPAibRA IVVSK-NH2 sequence into the C-terminus and combining it with fatty acid modification at the Lys site of the C-terminus, not only achieves efficient agonistic and biased signaling agonistic effects on GLP-1 and GIP receptors, but also significantly improves the stability of the polypeptide in vivo. Attached Figure Description

[0012] Figure 1The results show the detection of the biased signaling agonist effect of the polypeptide compound of the present invention on the human GLP-1 receptor (evaluated by β-arrestin-2 recruitment assay). Figure 2 The results show the detection of the biased signaling agonist effect of the polypeptide compound of the present invention on the human GIP receptor (evaluated by β-arrestin-2 recruitment assay). Figure 3 The results show the detection of the biased signaling agonist effect of the polypeptide compound of the present invention on the mouse GLP-1 receptor (evaluated by β-arrestin-2 recruitment assay). Figure 4 The results show the detection of the biased signaling agonist effect of the polypeptide compound of the present invention on the mouse GIP receptor (evaluated by β-arrestin-2 recruitment assay). Figure 5 The percentage change in body weight of the polypeptide compound of the present invention after 22 days of long-term administration to DIO mice; Figure 6 This is the blood glucose curve from an oral glucose tolerance test conducted on DIO mice after 22 days of long-term administration of the polypeptide compound of the present invention. Detailed Implementation

[0013] The technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of this application, and not all of the embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application. At the same time, in the description of the embodiments of this application, the terms "first," "second," etc., are only used to distinguish descriptions and should not be construed as indicating or implying relative importance. Thus, features defined with "first" and "second" may explicitly or implicitly include one or more features. In the description of the embodiments of this application, "multiple" means two or more, unless otherwise explicitly specified.

[0014] Example 1 Synthesis of SEQ ID NO: 1 The peptides in Example 1 were synthesized in glass peptide synthesis tubes using a fluorenylmethoxycarbonyl (Fmoc) / tert-butyl (tBu) solid-phase chemical synthesis method. Fmoc-Rink Amide-MBHA resin with a loading of 0.262 mmol / g was used as the solid-phase support, and standard protecting groups were used for all amino acid side chains. Lysine at position 40 was protected with Fmoc-Lys(Dde)-OH, and histidine at position 1 was protected with Boc-His(Trt)-OH. Before each coupling step, the Fmoc protecting groups on the resin were removed using 20% ​​piperidine / DMF (2 × 7 min). The coupling of all standard amino acids used a 4-molar excess of Fmoc-amino acids and HBTU / HOBT / DIPEA as condensing agents, with a coupling reaction time of 1–3 hours. After the peptide backbone synthesis was complete, the Dde protecting group on the 40th lysine residue was removed using 2% hydrazine hydrate / DMF solution (5 × 10 min). The 40th lysine side chain fatty acid side chain was coupled by introducing 2-[2-(2-Fmoc-amino-ethoxy)-ethoxy]-acetic acid (Fmoc-AEEA-OH), Fmoc-glutamic acid α-tert-butyl ester (Fmoc-Glu-OtBu), and mono-OtBu-octadecanoic acid in sequence. In each step, the Fmoc protecting group was removed using the same method as described above, along with a 4-molar excess of coupling reagent and HBTU / HOBT / DIPEA. The reaction was carried out for 3 hours.

[0015] After synthesis, the resin was thoroughly washed with dichloromethane, filtered, and air-dried. Then, using a TFA / anisole / phenol / EDT (volume ratio 90:5:3:2) cleavage buffer, the dried resin was transferred to a 10 mL reaction flask, 5 mL of the cleavage buffer was added, and the reaction was carried out at room temperature with shaking for 3 hours. After the reaction, the resulting reaction solution was poured into 10 volumes of ice-cold anhydrous diethyl ether, centrifuged to collect the precipitate, and washed three times with ice-cold anhydrous diethyl ether. Finally, it was air-dried at room temperature to obtain the crude peptide. The crude peptide was dissolved in a 50% methanol / 50% water mixture and purified by reversed-phase high-performance liquid chromatography (RP-HPLC) on a Shim-pack PREP-ODS(H) preparative column (250 × 20 mm, Shimadzu) using a 100% methanol / 0.1% TFA / water buffer system with a linear gradient of 40-80% methanol and a gradient time of 60 minutes. The purity of the target peptide was determined by analytical RP-HPLC, and fractions with a purity of 98% or higher were collected, lyophilized, and the final product was obtained. The molecular weight was determined by LC-MS. ESI-MS m / z: Calculated value [M+3H] 3+ 1686.9, [M+4H] 4+ 1265.5; Observation [M+3H] 3+ 1686.2, [M+4H] 4+1264.9.

[0016] Example 2 Determination of the agonistic activity of peptide compounds on human GLP-1 and GIP receptors The CHO cell line stably expressing the human GLP-1 receptor was cultured in F-12 medium containing 10% fetal bovine serum and 0.2 mg / mL Hygromycin B. The HEK293 cell line stably expressing the human GLP-1 receptor was cultured in DMEM medium containing 10% fetal bovine serum and 0.1 mg / mL Hygromycin B.

[0017] Following the kit instructions, prepare 1× Casein Buffer as the basal buffer. Serially dilute the test compound at specific ratios to prepare 10 different concentrations (1 nM, 0.2 nM, 0.04 nM, 0.008 nM, 0.0016 nM, 0.00032 nM, 0.000064 nM, 0.000013 nM, 0.0000026 nM, 0.00000051 nM). Each concentration of the compound is further diluted 10-fold with 1× Casein Buffer to the working concentration and thoroughly mixed before use. When cells reach 80% confluence, trypsin digestion is used to collect cells. Cells are then counted and seeded into 384-well plates at 9 μL per well. Next, add 1 μL of the 10-fold diluted working compound solution to each well. After centrifugation, incubate the 384-well plates at 37°C for 30 minutes. Subsequently, Eu-cAMP and ULight™-anti-cAMP antibodies were diluted to working concentrations with detection buffer, and 5 μL of each was added to each well. After mixing, the mixture was centrifuged and incubated at room temperature for 1 hour. After the reaction, the fluorescence signals of each well were detected sequentially at emission wavelengths of 665 nm and 620 nm using a microplate reader at an excitation wavelength of 330 nm. Finally, the experimental data were analyzed using GraphPad Prism 7.0 software, and the EC50 of each compound against human GLP-1 receptor and human GIP receptor was calculated using nonlinear regression. 50 The values ​​are shown in Table 1.

[0018] Table 1: EC50 values ​​(in nM) of peptide compounds for human GLP-1 and GIP receptors As shown in Table 1, the polypeptide compound of the present invention exhibits potent agonistic activity against both GLP-1 and GIP receptors. Its agonistic activity against GLP-1 is comparable to that of endogenous GLP-1 and approximately 12 times that of the commercially available drug tirzepatide. Simultaneously, the polypeptide's agonistic activity against GIP receptors is significantly higher than that of endogenous GIP and tirzepatide, with an increase of approximately 4-fold. These results clearly demonstrate that the polypeptide compound of the present invention is a dual agonist of both GLP-1 and GIP receptors with excellent activity.

[0019] Example 3 Determination of the biased signaling agonist effect of peptide compounds on human and mouse GLP-1 and GIP receptors (β-arrestin-2 recruitment assay) HEK293T cell lines (including human and mouse HEK293T-GCGR-β-Arrestin2, HEK293T-GIPR-β-Arrestin2, and HEK293T-GLP-1R-β-Arrestin2) were seeded into different types of culture plates: 96-well plates (30,000 cells per well, Opti-MEM medium), 384-well plates (10,000 cells per well, Opti-MEM medium), and 96-well plates (30,000 cells per well, Opti-MEM medium). All cells were cultured overnight at 37°C and 5% CO2.

[0020] Before the experiment, prepare a 1× concentration of Nano-Glo® Live Cell Substrate according to the kit instructions and aliquot it into each well (20 μL per well for a 96-well plate and 5 μL per well for a 384-well plate). Then, add the serially diluted test compound to each well (final concentration of 0.1% DMSO; 10 μL / well for a 96-well plate and 3 μL / well for a 384-well plate). After thorough and gentle mixing, incubate at room temperature for 10 minutes or in the dark for 15 minutes, depending on the cell type. Finally, detect the luminescence signal of each well using a BMG Labtech PHERAstar FSX multi-mode microplate reader.

[0021] like Figure 1 and Figure 2 As shown, the polypeptide compounds of this invention did not induce the recruitment of β-arrestin-2 when acting on human GLP-1 and GIP receptors. (Referring to the EC in Table 1) 50Data confirms that the polypeptide compound of the present invention exhibits significant signal-biased agonistic activity on both the GLP-1 and GIP receptors. The polypeptide compound of the present invention is a dual agonist possessing signal bias on both the GLP-1 and GIP receptors. In contrast, tirzepatide exhibits biased agonistic activity only on the GLP-1 receptor and no significant signal bias on the GIP receptor, consistent with previous reports (JCI Insight, 2020, 5, e140532).

[0022] In addition, such as Figure 3 and Figure 4 As shown, the peptide compounds of this invention exhibit significant biased agonistic effects not only on human GLP-1 and GIP receptors, but also on mouse GLP-1 and GIP receptors; while tirzepatide only shows biased agonistic activity on mouse GLP-1 receptors, with no significant signal bias on mouse GIP receptors. In summary, the above data fully demonstrate that the peptide compounds of this invention not only possess excellent GLP-1 and GIP receptor agonistic activity, but also simultaneously exhibit biased agonistic effects on both GLP-1 and GIP receptors, demonstrating great application potential and advantages as innovative dual GLP-1 / GIP receptor agonists.

[0023] Example 4 Pharmacokinetic properties of polypeptide compounds in rats Male SD rats were administered a subcutaneous (sc) injection of 100 nmol / kg, and blood samples were collected within 48 hours post-administration. Proteins were precipitated with acetonitrile, and plasma samples were analyzed by LC-MS. Pharmacokinetic parameters and half-life were calculated using WinonLin 5.2.1 (non-compartmental model), as shown in Table 2.

[0024] Table 2: Pharmacokinetic parameters of peptide compounds in rats As shown in Table 2, the peptide compounds of the present invention exhibit a significantly prolonged half-life in vivo, superior to existing once-weekly Tirzepatide. These results demonstrate that the peptide compounds of the present invention possess excellent pharmacokinetic properties supporting at least once-weekly dosing.

[0025] Example 5 Determination of gastrointestinal side effects of peptide compounds Male SD rats weighing 200–250 g were randomly divided into groups of eight and housed individually. For the first four days of the experiment, in addition to their normal rat diet, the rats were given a separate kaolin-based diet (Research Diets) placed in a separate compartment of the food funnel to allow them to acclimatize. The rats were fasted for 12 hours prior to the experiment. On day 1 (hour 0), the rats in each group were intraperitoneally injected with saline (blank), 100 nmol / kg tirzepatide, or 100 nmol / kg SEQ ID NO: 1, respectively. Following the injection, the rats were given either the weighed normal rat diet or the kaolin-based diet. At 24 hours, the rats in each group were again intraperitoneally injected with the same dose (saline, 100 nmol / kg tirzepatide, or SEQ ID NO: 1). The intake of normal rat diet and kaolin-based diet was recorded at 24 and 48 hours. The degree of gastrointestinal side effects induced by each compound was determined by analyzing the consumption of ordinary rat feed and kaolin feed.

[0026] Table 3: Food intake of SD rats in 24 hours: Normal rat diet and kaolin. Compared with the blank control group, P<0.001; ###: Compared with the Tirzepatide group, P<0.001 (One-Way ANOVA, Tukey post hoc test). Results are expressed as mean ± SD of 8 rats in each group.

[0027] Table 4: Food intake of SD rats in normal rat diet and kaolin clay at 48 hours Compared with the blank control group, P<0.001; ###: Compared with the Tirzepatide group, P<0.001 (One-Way ANOVA, Tukey post hoc test). Results are expressed as mean ± SD of 8 rats in each group.

[0028] As shown in Tables 3 and 4, the polypeptide compounds of the present invention exhibited significant and sustained appetite suppression in rats at both 24 and 48 hours, with significantly better appetite suppression effects than the tirzepatide control group. Simultaneously, the kaolin intake of rats treated with the polypeptide compounds of the present invention was comparable to that of the control group and significantly lower than that of the tirzepatide group, while the kaolin intake of rats in the tirzepatide group was significantly higher than that of the control group and the polypeptide compound group of the present invention. These results indicate that the polypeptide compounds of the present invention effectively suppress appetite without causing significant gastrointestinal side effects, and their safety is significantly better than that of tirzepatide. Therefore, the polypeptide compounds of the present invention possess both excellent efficacy and low adverse reactions in disease treatment, demonstrating greater application value and promising prospects for promotion.

[0029] Example 6 Effects of peptide compounds on body weight, blood glucose, and blood lipids in diet-induced obese (DIO) mice Male C57BL / 6J mice weighing approximately 22 g were selected and fed a high-fat diet (D12492, Research Diets) for approximately 20 weeks to establish a diet-induced obesity (DIO) mouse model. Mice weighing over 45 g were selected for subsequent experiments. DIO mice were divided into groups of 8 mice each. The control group received subcutaneous injections of saline (10 mL / kg), while the treatment groups received subcutaneous injections of either tirzepatide (10 nmol / kg) or SEQ ID NO: 1 (10 nmol / kg), administered every 2 days. Mouse weight was recorded every two days during the experiment. On day 23, all mice underwent an oral glucose tolerance test (OGTT): after an 8-hour overnight fast, 1.5 g / kg glucose was administered orally. Tail vein blood samples were collected at 0, 15, 30, 60, and 120 minutes to measure blood glucose levels and assess changes in glucose tolerance. After the OGTT, all groups of mice rested for one day. On day 24 of the experiment, blood samples were collected from mice in a non-fasting state to prepare serum, and the serum triglyceride and cholesterol levels were measured.

[0030] Table 5: Changes in body weight and glucose tolerance in DIO mice during a 22-day dosing period. Compared with the blank control group, P<0.001; ###: Compared with the tirzepatide group, P<0.001. Results are expressed as mean ± SD of 8 mice in each group.

[0031] like Figure 5 , Figure 6As shown in Table 5, the polypeptide compound of the present invention, after continuous administration to DIO mice for 22 days, significantly reduced the weight of the mice, and the weight loss effect was significantly better than that of tirzepatide. Furthermore, the OGTT results after treatment showed that the blood glucose level and blood glucose AUC of the mice in the polypeptide compound group were significantly lower than those in the tirzepatide group, indicating that its effect on improving glucose tolerance was also more prominent. In summary, the polypeptide compound of the present invention exhibits excellent efficacy in both lowering blood glucose and reducing weight.

[0032] Table 6: Serum triglyceride and cholesterol levels in DIO mice after 22 days of treatment Compared with the blank control group, P<0.001; ###: Compared with the tirzepatide group, P<0.001. Results are expressed as mean ± SD of 8 mice in each group.

[0033] As shown in Table 6, the polypeptide compound of the present invention, after being administered to DIO mice for 22 consecutive days, significantly reduced the serum triglyceride and cholesterol levels in the mice, and its effect on reducing serum lipids was significantly better than that of tirzepatide.

[0034] Obviously, those skilled in the art can make various modifications and variations to the embodiments of the present invention without departing from the spirit and scope of the invention. Therefore, if these modifications and variations fall within the scope of the claims of the present invention and their equivalents, the present invention also intends to include these modifications and variations.

Claims

1. A polypeptide or a pharmaceutically acceptable salt thereof having biased agonistic activity towards GLP-1 and GIP receptors, characterized in that, The general formula of the amino acid sequence of the polypeptide is: His-Xaa1-Glu-Gly-Thr-Tyr-Thr-Asn-Asp-Val-Ser-Ile-Xaa2-Leu-Asp-Lys-Ile-Ala-Gln-Lys-Al a-Phe-Val-Gln-Trp-Leu-Ile-Ala-Gly-Gly-Pro-Pro-Xaa3-Arg-Ala-Ile-Val-Val-Ser-Xaa4-NH2; in, Xaa1 is derived from Ala or Aib; Xaa2 is derived from Ile or Aib; Xaa3 is derived from Pro or Aib; Xaa4 is derived from Lys or Lys with modified side chains; The structural formula of the Lys with modified side chains is shown below: 。 2. The polypeptide with biased agonistic activity towards GLP-1 and GIP receptors according to claim 1, characterized in that, The amino acid sequence structure of the polypeptide is as follows: 。 3. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the polypeptide of any one of claims 1-2 and a pharmaceutically acceptable carrier, diluent or excipient.

4. Use of the polypeptide having GLP-1 receptor and GIP receptor biased agonist activity as described in claim 1 or 2, or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition as described in claim 3, in the preparation of a medicament for treating metabolic diseases or conditions.

5. The use according to claim 4, characterized in that, The metabolic diseases or conditions mentioned include diabetes, obesity, and dyslipidemia.

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