Long-acting polypeptide analogue and application thereof

Abstract

The invention discloses a long-acting polypeptide analogue and application thereof.The polypeptide analogue can target a GLP-1 receptor, a GIP receptor and an NPY2 receptor at the same time and has multiple biological functions, the advantages of GLP-1 in diabetes treatment can be brought into play, the positive effects of GIP in glycometabolism and lipid metabolism regulation and appetite inhibition are achieved, and the polypeptide analogue can be used for treating diabetes mellitus and treating diabetes mellitus and treating diabetes mellitus and treating diabetes mellitus and treating diabetes mellitus and treating diabetes mellitus and treating diabetes mellitus and treating diabetes mellitus. The effect of the PYY3-36 on regulating and controlling the appetite is fused. The multi-target synergistic effect is beneficial to optimization of sugar, fat and energy balance. The polypeptide analogue shows a wider application prospect in the field of preparation of drugs for preventing and treating metabolic syndromes, such as diabetes, obesity and other diseases.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a long-acting polypeptide analog and its applications. Background Technology

[0002] Weight control is crucial for improving metabolic health and can effectively delay the development of various obesity-related complications, including type 2 diabetes mellitus (T2DM). Currently used hypoglycemic drugs, such as sodium-glucose cotransporter 2 (SGLT2) inhibitors and glucagon-like peptide-1 (GLP-1) receptor agonists, can promote weight loss to some extent, but their efficacy varies. For severely obese patients, bariatric surgery remains the most effective treatment; however, it carries certain risks and is therefore not the preferred first-line treatment. In recent years, tirzepatide, a novel drug with both glucose-dependent insulinotropic peptide (GIP) receptor and GLP-1 receptor agonist activity, has shown greater weight loss efficacy in T2DM patients than single GLP-1 receptor agonists. Despite these positive advancements, there is still a pressing need to develop more effective weight-loss drugs for weight management in obese and T2DM patients.

[0003] Notably, in vitro studies show that telposide's effect on the GIP receptor is similar to that of endogenous GIP, while its signal transduction on the GLP-1 receptor exhibits certain unique characteristics. Specifically, when telposide activates the GLP-1 receptor, it tends to promote the production of cyclic adenosine monophosphate (cAMP) while its recruitment of β-arrestin is relatively weak. This biased signal transduction helps the GLP-1 receptor remain more on the cell membrane, thereby enhancing the drug's efficacy. Furthermore, studies have shown that reducing β-arrestin recruitment helps GLP-1 receptor agonists achieve better blood glucose and weight loss effects. Therefore, for GLP-1 receptor agonists, biased activation of the GLP-1 receptor (i.e., more inclined to induce cAMP production and less recruiting β-arrestin) is expected to lead to better efficacy.

[0004] PYY 1-36 Both PYY and GLP-1 are secreted by intestinal L cells in tandem after eating. 1-36 It will then be rapidly cleaved into PYY by dipeptidyl peptidase-4 (DPP-IV). 3-36 Compared to other NPY receptor subtypes (such as NPY1, NPY4, and NPY5 receptors), PYY... 3-36 It exhibits stronger selectivity for the NPY2 receptor. By acting on the NPY2 receptor, PYY... 3-36It can effectively suppress appetite and reduce food intake. Studies have already indicated that long-acting PYY... 3-36 Analogs outperform natural PYY in weight control. 3-36 Furthermore, animal experiments showed that PYY 3-36 Combined use with exendin-4, oxyntomodulin, or GLP-1 can enhance overall efficacy. Therefore, designing multi-target peptides with both GLP-1 and NPY2 receptor agonist effects has broad development value and application potential.

[0005] In summary, multi-target peptides that simultaneously activate GLP-1, GIP, and NPY2 receptors hold promise for synergistically regulating appetite, promoting insulin secretion, and improving metabolic disorders, thereby comprehensively enhancing hypoglycemic and weight-loss effects. Furthermore, if these compounds exhibit signal transduction bias when activating the GLP-1 receptor—that is, a stronger induction of cAMP production and weaker recruitment of β-arrestin—it would not only enhance efficacy but also help reduce adverse reactions associated with GLP-1 receptor agonists, significantly improving safety and tolerability. Therefore, developing novel peptide drugs with triple agonist activity against GLP-1, GIP, and NPY2 receptors, and selectively regulating GLP-1 receptor signal transduction, holds promise for providing more efficient and less side-effect-prone innovative solutions for the treatment of metabolic diseases such as obesity and type 2 diabetes mellitus (T2DM). Summary of the Invention

[0006] This invention provides a long-acting peptide analog and its applications. This peptide analog can simultaneously target GLP-1, GIP, and NPY2 receptors, possessing multiple biological functions: it not only leverages the advantages of GLP-1 in diabetes treatment but also exhibits the positive effects of GIP in regulating glucose and lipid metabolism and suppressing appetite, and incorporates NPY2 receptors. 3-36 Effects on appetite regulation. The multi-target synergistic effect helps optimize glucose, lipid, and energy balance. This peptide analog shows broad application prospects in the development of drugs for the prevention and treatment of metabolic syndromes such as diabetes and obesity.

[0007] To achieve the above-mentioned objectives, the technical solution of the present invention is as follows: A long-acting peptide analog, wherein the amino acid sequence (SEQ ID NO: 1) of the peptide analog is: .

[0008] The present invention also provides a pharmaceutical composition comprising a long-acting polypeptide analog, comprising the aforementioned long-acting polypeptide analog and a pharmaceutically acceptable carrier, diluent or excipient.

[0009] The present invention also provides the use of the above-mentioned long-acting peptide analog or pharmaceutical composition of the above-mentioned long-acting peptide analog in the preparation of a medicament for treating metabolic diseases or conditions, including diabetes and obesity.

[0010] Compared with the prior art, the beneficial technical effects of the present invention are as follows: (1) The long-acting peptide analogue of the present invention can synergistically act on GLP-1 receptor, GIP receptor and NPY2 receptor, and has the combined effects of GLP-1, GIP and NPY2 receptors. 3-36 This analogue possesses multiple biological activities. It not only effectively leverages the hypoglycemic and diabetes-treating effects of GLP-1, but also retains the advantages of GLP-1 in regulating glucose and lipid metabolism and suppressing appetite, while fully utilizing PYY... 3-36 The high selectivity for the NPY2 receptor achieves a significant appetite-suppressing effect. Through the synergistic effect of multiple targets, the peptide analogs of this invention can comprehensively regulate glucose metabolism, lipid metabolism, and energy metabolism, showing great application potential and broad market prospects in the prevention and treatment of metabolic syndromes (such as diabetes and obesity-related diseases).

[0011] (2) The peptide analogs of the present invention, in addition to achieving synergistic regulation of multiple targets, also possess selective agonistic ability towards the GLP-1 receptor, exhibiting a unique signal transduction bias. Specifically, when activating the GLP-1 receptor, the compound tends to induce cAMP production, while its recruitment effect on β-arrestin is weak. This bias in signal transduction pathways helps to enhance drug efficacy, achieve better hypoglycemic and weight-loss effects, and reduce adverse reactions mediated by β-arrestin, thereby significantly improving drug safety and tolerability. With this innovation, the present invention provides strong technical support for the development of more efficient, safer, and less side-effect-prone drugs for the treatment of metabolic diseases, and also brings new solutions and application prospects for the treatment of related diseases. Attached Figure Description

[0012] Figure 1 The results show the detection of the biased signaling agonist effect of the polypeptide analog of the present invention on the human GLP-1 receptor (evaluated by β-arrestin-2 recruitment experiment).

[0013] Figure 2 The percentage change in body weight of the polypeptide analog of the present invention after 22 days of long-term administration to DIO mice.

[0014] Figure 3 The glucose tolerance test curves of the polypeptide analog of the present invention were obtained in DIO mice after long-term administration for 22 days. Detailed Implementation

[0015] The present invention will now be described in detail with reference to the accompanying drawings and embodiments.

[0016] 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.

[0017] Furthermore, the amino acid sequence of the polypeptide analog described in this invention includes amino acid single-letter codes (e.g., His = H) commonly used in this invention, as well as other non-natural amino acids, such as α-aminoisobutyric acid (Aib). The structures of non-natural amino acids are described below: .

[0018] Furthermore, unless otherwise specified, all amino acid residues in the polypeptides of the present invention are preferably in the L configuration.

[0019] Furthermore, the "-NH2" portion at the C-terminus of the sequence indicates an amide group (-CONH2) at the C-terminus.

[0020] 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.

[0021] Example 1 Synthesis of SEQ ID NO: 1 The synthesis of the polypeptide analogs of this invention employs an orthogonal protected solid-phase synthesis method using Fmoc / tBu. Fmoc-RinkAmide-MBHA resin (loading 0.356 mmol / g) is used as the carrier. 0.281 g of the resin is fully swollen and washed with dichloromethane and DMF, followed by deprotection of the Fmoc protecting group with 20% piperidine / DMF. Amino acids are sequentially linked from the C-terminus to the N-terminus using HBTU / HOBT / DIPEA as condensing agents, with Fmoc deprotection performed at each step. The Lys group at position 20 is Fmoc-Lys(Dde)-OH, with Dde deprotection using 2% hydrazine hydrate / DMF. Side chain modification involves the sequential linkage of two molecules of Fmoc-AEEA-OH, Fmoc-Glu-OtBu, and tert-butyl octadecanoate. Both main chain and side chain linkages follow the same condensation and deprotection steps. After synthesis, the resin was washed with dichloromethane and dried. The peptide chain was cleaved using TFA / anisole / phenol / EDT (90:5:3:2, v / v), precipitated with ice-cold diethyl ether, washed, and dried to obtain crude peptide. The crude peptide was purified by preparative HPLC gradient purification (0.1% TFA / water-methanol system). After collecting the target peak, the peptide was rotary evaporated and lyophilized to obtain pure peptide. Analytical HPLC showed a purity >98%, and the molecular weight determined by mass spectrometry was consistent with the theoretical value, indicating successful synthesis and purification. The theoretical relative molecular mass is 6610.6. ESI-MS m / z: Calculated value [M+5H] 5+ 1323.1, [M+6H] 6+ 1102.8; Observation [M+5H] 5+ 1322.3, [M+6H] 6+ 1101.9.

[0022] Example 2 Assay of the agonistic activity of peptide analogs against GLP-1, GIP, NPY1, NPY2, NPY4, and NPY5 receptors To evaluate the agonistic activity of peptide analogs on GLP-1 and GIP receptors, HEK293 cells stably expressing the target receptors were first constructed, and functional assays were performed using a cAMP signal monitoring system. The peptide compound was diluted 10 times (4-fold) with DMSO as the solvent. Cell suspensions were then prepared and harvested by centrifugation at 1000 rpm for 5 minutes at room temperature. The cells were resuspended in HBSS buffer, and cell density and viability were measured. Cells were finally resuspended at 200,000 cells / mL in assay buffer, and 10 μL was transferred to OptiPlate-384 microplates. 100 nL of the compound was transferred to the corresponding well using an Echo system, briefly centrifuged (1000 rpm, 5 seconds), and incubated at room temperature for 30 minutes. Then, the probe reagent was added, and the plates were sealed with a TopSeal-A membrane. After incubation at room temperature for 1 hour, the sealing membrane was removed, and the signal was read using an EnVision instrument. EC50 was calculated using GraphPad Prism 7.0 software. 50 value.

[0023] To detect the agonistic effects of peptide compounds on NPY receptors (human NPY1, NPY2, NPY4, and NPY5), HEK-293 cells stably co-expressing Gα15 protein were used. Cells were seeded at 20 μL / well (20,000 cells / well) into 384-well plates and incubated overnight at 37°C with 5% CO2. After discarding the culture medium, 20 μL of FLIPR buffer and 20 μL of Fluo-4Direct dye (2-fold concentration) were added to each well, and the plates were incubated at 37°C for 50 minutes, followed by incubation at room temperature for 10 minutes. The peptides were also diluted 4-fold at 10 concentrations, and 750 nL of the compound was transferred to each well using an Echo transducer. 30 μL of FLIPR assay buffer was added, and then 10 μL of the compound was transferred to each well using a FLIPR Tetra system (Molecular Devices), with fluorescence monitored in real time (Ex 494 nm / Em 516 nm). The obtained EC50 values ​​were also analyzed using GraphPad Prism 7.0.

[0024] Table 1: EC5 activity of peptide analogs on GLP-1 receptor, GIP receptor, NPY1 receptor, NPY2 receptor, NPY4 receptor, and NPY5 receptor 50 Value (represented by nM) As shown in Table 1, the peptide analogs of the present invention exhibit potent agonistic activity against GLP-1, GIP, and NPY2 receptors. Notably, these peptide analogs demonstrate high receptor selectivity within the NPY receptor family, with significantly better agonistic activity against NPY2 receptors than against NPY1, NPY4, and NPY5 receptors, and their selectivity for NPY2 receptors is significantly higher than that for PY2 receptors. 3-36 Selective activation of the NPY2 receptor not only enhances the expected pharmacological effects but also minimizes the associated side effects of increased appetite caused by activation of NPY1 / NPY5 receptors, as well as the inhibition of gallbladder emptying and pancreatic enzyme secretion mediated by the NPY4 receptor (J. Med. Chem. 2018, 61, 23, 10519–10530). Therefore, the peptide analogs of this invention exhibit significant advantages in both safety and efficacy, and have broader prospects for clinical application.

[0025] Example 3 Determination of the biased signaling agonist effect of peptide analogs on GLP-1 receptor (β-arrestin-2 recruitment assay) HEK293T-GLP-1R-β-Arrestin2 cell lines were seeded into 96-well plates (30,000 cells per well, in Opti-MEM medium). All cells were cultured overnight at 37°C with 5% CO2. Before the experiment, 1× Nano-Glo® Live Cell Substrate was prepared according to the kit instructions and aliquoted into each well (20 μL per well for 96-well plates). Then, serially diluted test compounds were added to each well (final concentration of 0.1% DMSO; 10 μL / well for 96-well plates). After thorough and gentle mixing, the cells were incubated at room temperature for 10 minutes or in the dark for 15 minutes, depending on the cell type. Finally, the luminescence signal of each well was detected using a BMG Labtech PHERAstar FSX multi-mode microplate reader.

[0026] like Figure 1 As shown, the polypeptide compounds of this invention did not induce the recruitment of β-arrestin-2 when acting on the GLP-1 receptor. (See Table 1 for EC...) 50 The data confirm that the polypeptide compounds of the present invention all have significant signal-biased agonistic effects on the GLP-1 receptor.

[0027] Example 4 Gastrointestinal side effects assay of peptide analogs Male SD rats weighing 200–250 g were randomly divided into several groups of 8 rats each, housed individually. Four days prior to the experiment, in addition to their regular diet, rats were provided with kaolin-based research diets, placed separately in food funnel compartments to help them acclimatize to the presence of kaolin. All rats were fasted for 12 hours before the experiment. On day 1 (hour 0), each group of rats was intraperitoneally injected with physiological saline (blank control), 100 nmol / kg tirzepatide, or 100 nmol / kg SEQ ID NO: 1, respectively. After injection, each rat was provided with pre-weighed regular and kaolin-based diets. Twenty-four hours into the experiment, the intraperitoneal injection was repeated, with consistent dosage and treatment across all groups. During the experiment, the intake of regular and kaolin-based diets was recorded at 48 hours. The data on feed consumption was analyzed to assess the severity of gastrointestinal side effects induced by the different treatment compounds.

[0028] Table 2: Food intake of SD rats in normal rat diet and kaolin clay at 48 hours sample Feed intake of ordinary rat feed (g) Kaolin intake (g) Blank control 42.4±2.2 g 0.61±0.09 g Tirzepatide <![CDATA[26.2±0.9 g *** ]]> <![CDATA[1.64±0.11 g *** ]]> SEQ ID NO: 1 <![CDATA[12.6±0.5 g ***,### ]]> <![CDATA[0.56±0.04 g ### ]]> *** 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.

[0029] As shown in Table 2, the peptide analogs of the present invention exhibited significant and sustained appetite-suppressing effects in animal experiments, with a significantly better appetite reduction effect than tirzepatide. More importantly, the kaolin intake of rats in the peptide analog group was almost the same as that in the control group and significantly lower than that in the tirzepatide-treated group, while the kaolin intake of rats in the tirzepatide group was significantly higher than that in the other groups. These data fully demonstrate that the peptide analogs of the present invention achieve excellent appetite suppression without causing significant gastrointestinal side effects, and their safety is far superior to that of the existing drug tirzepatide.

[0030] Regarding its mechanism of action, tirzepatide non-selectively activates GIP receptors and selectively activates GLP-1 receptors (JCI Insight, 2020, 5, e140532). The peptide analog of this invention not only possesses GIP receptor agonist activity comparable to tirzepatide and a highly selective activation ability of GLP-1 receptors, but also achieves synergistic activation of NPY2 receptors. It is worth emphasizing that, through synergistic activation of NPY2 receptors and selective activation of GLP-1 receptors, the peptide of this invention not only does not increase gastrointestinal side effects, but also effectively alleviates the common gastrointestinal adverse reactions of traditional GLP-1 receptor agonists due to their lack of selective activation. This unique molecular mechanism of action not only achieves more potent appetite suppression but also significantly improves safety and greatly reduces the risk of side effects. In summary, the peptide analog of this invention has significant and unique advantages in terms of innovation and safety.

[0031] Example 5 Effects of peptide analogs on body weight and blood glucose in diet-induced obese (DIO) mice Male C57BL / 6J mice weighing approximately 20 g were selected and fed a high-fat diet (D12492, Research Diets) for at least 16 weeks to establish a DIO mouse model. Mice weighing more than 45 g and with similar initial weights in each group were selected and divided into groups of 8. The control group was given saline (10 mL / kg), while the experimental groups were given tirzepatide (10 nmol / kg) and SEQ ID NO: 1 (10 nmol / kg), respectively, administered subcutaneously every other day. Throughout the experiment, mouse weight was measured and recorded every two days. On day 22 of drug administration, an oral glucose tolerance test (OGTT) was performed. All mice were fasted for 8 hours the previous night and then administered glucose by gavage at a dose of 1.5 g / kg. Blood samples were collected at 0, 15, 30, 60, and 120 minutes to measure blood glucose levels and assess glucose tolerance.

[0032] Table 3: Changes in body weight and glucose tolerance in DIO mice during a 22-day dosing period. Sample (dosage) Changes in body weight (%) of mice in each group on day 22 The area under the blood glucose curve (AUC) values ​​of mice in each OGTT group after treatment. Blank control group (saline group) 1.3±2.7 2185±112 Tirzepatide (10 nmol / kg) <![CDATA[−19.2±1.9 *** ]]> <![CDATA[1530±79 *** <!-- 5 -->]]> SEQ ID NO: 1 (10 nmol / kg) <![CDATA[−31.9±1.6 ***,### ]]> <![CDATA[714±58 ***,### ]]> *** 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] like Figure 2 , Figure 3As shown in Table 3, the polypeptide analogs of the present invention exhibit significantly better effects than tirzepatide in both weight loss and improved glucose tolerance, fully demonstrating that the polypeptide analogs of the present invention have excellent therapeutic effects in lowering blood sugar and reducing weight.

Claims

1. A long-acting polypeptide analog, characterized in that, The amino acid sequence of the polypeptide analogue is: 。 2. A pharmaceutical composition comprising a long-acting polypeptide analogue, characterized in that, The pharmaceutical composition comprises the polypeptide analogue of claim 1 and a pharmaceutically acceptable carrier, diluent or excipient.

3. Use of a polypeptide analogue of claim 1 or a pharmaceutical composition of claim 2 in the manufacture of a medicament for the treatment of a metabolic disease or disorder.

4. Use according to claim 3, characterized in that, The metabolic disease or disorder is diabetes and obesity.

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