Agonistic polypeptides targeting glp-1r / gcgr / gipr, polypeptide derivatives and their preparation and use

Abstract

The application discloses an agonistic activity polypeptide, a polypeptide derivative and a preparation and application thereof targeting GLP-1R / GCGR / GIPR, an amino acid sequence of the polypeptide is shown as SEQ ID NO. 1-3, and the C terminal is in an amidation form. The polypeptide derivative is prepared by replacing the second amino acid at the N terminal with Aib and connecting a lipidized side chain to a specific lysine side chain on the basis of the polypeptide, and the C terminal is in a carboxylic acid form, and a sequence thereof is shown as SEQ ID NO. 4-6. The application further provides a preparation method of the polypeptide and the polypeptide derivative. The polypeptide disclosed by the application has clear triple agonistic activity to GLP-1R, GCGR and GIPR, the polypeptide derivative still retains agonistic activity to at least one receptor, can be used as a GLP-1R, GCGR, GIPR or GLP-1R / GCGR / GIPR agonist, and can be used for preparing a medicine or a pharmaceutical composition for preventing or treating a glycolipid metabolism abnormality disease.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to agonistic active peptides targeting GLP-1R / GCGR / GIPR, peptide derivatives, and their preparation and application. Background Technology

[0002] In recent years, drug development based on the incretin system has become an important direction in the treatment of metabolic diseases. Glucagon-like peptide-1 receptor (GLP-1R), glucagon receptor (GCGR), and glucose-dependent insulinotropic peptide receptor (GIPR) play crucial roles in glycemic homeostasis regulation, pancreatic islet function maintenance, energy metabolism, and weight control. Multi-target agonists that simultaneously activate multiple of these receptors are expected to produce synergistic effects in lowering blood glucose, weight loss, and overall metabolic improvement. Therefore, the development of dual or triple agonists of GLP-1R, GCGR, and GIPR has become a research hotspot.

[0003] In existing technologies, some multi-target agonist peptides based on natural peptide hormones have entered the clinical application or research stage. Early developments mainly focused on single-target GLP-1R agonists, followed by the development of GIPR / GLP-1R dual-target agonists. For example, telpolide is obtained by amino acid substitution, introduction of non-natural amino acids, C-terminal amidation, and fatty chain modification based on the GIP sequence. Furthermore, to accommodate multiple metabolic regulatory effects, researchers have proposed a GLP-1R / GIPR / GCGR triple agonist strategy; for example, peptide 20 and retatrutide have been reported as triple agonist peptides. These molecules are typically based on natural incretin or glucagon-related sequences, constructed by retaining key active fragments and performing amino acid site substitutions, extensions, truncations, or chemical modifications. They are prepared using solid-phase synthesis, and cAMP levels and pEC are detected using cell models expressing the corresponding receptors. 50 To evaluate stimulatory activity.

[0004] However, existing technologies still have significant limitations. On the one hand, the number of publicly disclosed peptide molecules with triple agonist activity of GLP-1R, GCGR, and GIPR is still very limited, resulting in an insufficient reserve of novel candidate molecules that can serve as the basis for subsequent drug development. On the other hand, existing molecular designs mostly revolve around known natural peptide backbones, limiting the space for novel sequence types and structural innovation. Furthermore, GLP-1R, GCGR, and GIPR are all class B G protein-coupled receptors, and even small changes in the peptide sequence can significantly affect multi-receptor binding and signal transduction. Achieving effective and balanced agonist activity of all three receptors simultaneously on the same peptide is technically challenging. Currently, only peptides that have been validated in vitro to induce clear changes in cAMP signaling at the cellular level and exhibit corresponding pEC responses are readily available. 50 Characteristic triple agonist molecules are still lacking. Summary of the Invention

[0005] The technical problem to be solved by this invention is to provide a series of novel peptides and their derivatives that can simultaneously exert agonistic effects on GLP-1R, GCGR, and GIPR, and exhibit clear triple agonistic activity in in vitro cell experiments, as evidenced by changes in cAMP levels and pEC. 50 This will serve as an evaluation metric to address the shortcomings of existing technologies, such as the lack of novel triple-agonist active peptides and the limited balance of receptor activity, thereby providing new candidate active molecules for the development of drugs related to metabolic diseases.

[0006] To achieve the above objectives, the present invention provides the following technical solution: This invention provides an agonistic active polypeptide targeting GLP-1R / GCGR / GIPR, the amino acid sequence of which is shown in SEQ ID NO.1~3: SEQ ID NO.1: HAQGTFTSDYSKYLDEQAAKEFIAWLVET-NH2; SEQ ID NO.2: HAQGTFTSDYSKYLDKRAQAAFVQWLLNG-NH2; SEQ ID NO. 3: HSQGTFTSDYSKYLDKIAAAEFVQWLLDG-NH2.

[0007] This invention also provides a polypeptide derivative, which is obtained by structural modification of an agonistic polypeptide targeting GLP-1R / GCGR / GIPR. The structural modification includes replacing the second amino acid at the N-terminus of the parent polypeptide with Aib and attaching an esterified side chain at a specific lysine site. The amino acid sequence of the polypeptide derivative is shown in SEQ ID NO. 4~6. SEQ ID NO.4: H-Aib-QGTFTSDYSKYLDEQAAK(AEEA-AEEA-γGlu-C18-diacid)EFIAWLVET-OH; SEQ ID NO.5: H-Aib-QGTFTSDYSKYLDK(AEEA-AEEA-γGlu-C18-diacid)RAQAAFVQWLLNG-OH; SEQ ID NO.6: H-Aib-QGTFTSDYSKYLDK(AEEA-AEEA-γGlu-C18-diacid)IAAAEFVQWLLDG-OH; In this designation, Aib is α-aminoisobutyric acid, AEEA is a hydrophilic linker, γGlu is a γ-glutamyl group, and C18-diacid is a C18 diacid aliphatic chain group.

[0008] Preferably, in the polypeptide derivative, Aib can be replaced with other non-natural amino acids that can enhance conformational stability, and the AEEA-AEEA-γGlu-C18-diacid esterified side chain can be replaced with other pharmaceutically acceptable aliphatic chain modification groups or linkers.

[0009] The present invention also provides a method for preparing the above-mentioned polypeptide using the Fmoc solid-phase polypeptide synthesis method, specifically including the following steps: S1. The solid support Rink Amide-MBHA Resin resin was swollen with dichloromethane and then washed with DMF; S2. Remove the Fmoc protecting group on the resin using Fmoc deprotection solution, and then couple the corresponding Fmoc protecting amino acids sequentially from the C-terminus to the N-terminus according to the target polypeptide sequence. S3. After the coupling is completed, check to confirm whether the coupling reaction is complete. If the coupling is incomplete, repeat the coupling steps. S4. After the main chain is assembled, remove the terminal Fmoc protecting group and wash the resin with DMF, dichloromethane and methanol in sequence. S5. Add lysis buffer to the resin to lyse the target peptide from the resin and remove the side chain protecting groups. After lysis, filter the solution, collect the filtrate, and add cold ether to precipitate the peptide. The precipitate is then centrifuged, washed with ether, and dried to obtain the crude peptide. S6. After purification by reversed-phase high-performance liquid chromatography, the main peak component was collected, and after analysis to confirm that the purity was qualified, it was freeze-dried to obtain the target peptide.

[0010] Preferably, the Fmoc protected amino acids in step S2 include, but are not limited to, at least one of: Fmoc-His(Trt)-OH, Fmoc-Ala-OH, Fmoc-Ser(tBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Gly-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Ile-OH, Fmoc-Trp(Boc)-OH, Fmoc-Leu-OH, and Fmoc-Val-OH.

[0011] Preferably, the detection in step S3 can be performed using conventional solid-phase synthesis detection methods, including but not limited to ninhydrin detection and the Kaiser test.

[0012] Preferably, the lysis solution in step S5 is a trifluoroacetic acid system.

[0013] Preferably, the purification in step S6 uses an acetonitrile / water system as the mobile phase, and a gradient elution is set according to the retention behavior of the target peptide.

[0014] The present invention also provides a method for preparing the above-mentioned polypeptide derivative, which uses the Fmoc solid-phase polypeptide synthesis method and specifically includes the following steps: S1. Select a solid support based on the C-terminal amino acid of the target polypeptide; S2. Assemble the derivative backbone sequentially from C-terminus to N-terminus according to the target sequence, wherein Fmoc-Aib-OH is coupled at the second position corresponding to the N-terminus of the parent polypeptide, and then Fmoc-His(Trt)-OH is coupled at the N-terminus. S3. After the main chain is assembled, the terminal Fmoc protecting group is removed to obtain the polypeptide main chain containing Aib at the N-terminus. S4. Selectively remove the protecting group of the target lysine side chain and couple the AEEA-AEEA-γGlu-C18-diacid modifying group onto the target lysine side chain; S5. After modification, wash the resin, add lysis buffer to the resin to lyse and remove side chain protecting groups. The lysis product is precipitated with cold diethyl ether, centrifuged, washed and dried to obtain crude peptide. S6. After purification by reversed-phase high-performance liquid chromatography, the main peak component was collected, and after analysis to confirm that the purity was qualified, it was freeze-dried to obtain the target polypeptide derivative.

[0015] Preferably, the selection in step S1 specifically involves: when the target peptide's C-terminus is Thr, using Fmoc-Thr(tBu)-WangResin as a solid-phase support; when the target peptide's C-terminus is Gly, using Fmoc-Gly-Wang Resin as a solid-phase support.

[0016] Preferably, in step S2, for lysine residues that require side-chain esterification modification, a selectively removable side-chain protecting group, including but not limited to Mtt and ivDde, is used for protection; for lysine residues that do not participate in esterification modification, a Boc protecting group is used for protection.

[0017] Preferably, the AEEA-AEEA-γGlu-C18-diacid modifying group in step S4 can be coupled as a pre-synthesized whole fragment, or it can be introduced by gradually coupling AEEA, AEEA, γGlu and C18-diacid.

[0018] The present invention also provides the use of the above-mentioned polypeptide or polypeptide derivative as one or more receptor agonists of GLP-1R, GCGR, and GIPR.

[0019] Preferably, the polypeptide has triple agonistic activity against GLP-1R / GCGR / GIPR; the polypeptide derivative has agonistic activity against at least one of the receptors GLP-1R, GIPR, and GCGR.

[0020] Preferably, the application is for the preparation of agonist drugs of GLP-1R, GCGR, GIPR or GLP-1R / GCGR / GIPR.

[0021] Furthermore, the GLP-1R, GCGR, GIPR, or GLP-1R / GCGR / GIPR agonist drugs include drugs for the prevention and treatment of disorders of glucose and lipid metabolism; the disorders of glucose and lipid metabolism include, but are not limited to, obesity, type 2 diabetes, impaired glucose tolerance, insulin resistance, and metabolic syndrome.

[0022] The present invention also provides a pharmaceutical composition comprising the above-mentioned polypeptide or polypeptide derivative.

[0023] Preferably, the pharmaceutical composition includes an injection solution, a lyophilized powder for injection, a sterile powder for injection, a sustained-release formulation, or a long-acting formulation.

[0024] The present invention has the following beneficial effects: 1) This invention provides a new set of polypeptide sequences and their derivatives, which expands the candidate molecular range of GLP-1R / GCGR / GIPR multi-receptor agonist active polypeptides, enriches the reserve of active molecules in the field of triple agonist peptides, and provides a new material basis for subsequent drug development. 2) The polypeptides provided by this invention have a novel sequence type, and their signal levels in the three receptor systems are significantly higher than those of the DMSO blank control. Furthermore, they all exhibit measurable pEC values ​​on GLP-1R, GCGR, and GIPR. 50 The value has been clearly demonstrated through in vitro functional experiments to have multi-receptor agonistic potential and clear triple agonistic activity against GLP-1R, GCGR and GIPR; 3) The peptides provided by this invention exhibit differentiated activity characteristics on different receptors and have different receptor activity spectra. They can show high agonistic activity on GLP-1R, GCGR and / or GIPR respectively, which can provide a greater selection space for screening advantageous molecules according to different research and development needs. 4) The peptide provided by this invention exhibits competitive in vitro agonistic activity on specific receptors compared to existing representative triple agonists. The pEC of the peptide corresponding to SEQ ID NO.1 on GLP-1R is shown.50 The pEC values ​​of the peptide corresponding to SEQ ID NO.3 on GLP-1R and GCGR are higher than those of retatrutide. 50 It is also higher than retatrutide, indicating that the novel polypeptide provided by this invention not only has multi-receptor agonist ability, but also exhibits strong in vitro functional activity on some key receptors. 5) This invention not only provides peptides, but also provides derivatives obtained by further modification of peptides. Some derivatives retain agonistic activity against one or more target receptors after modification. For example, the derivative of SEQ ID NO.2, SEQ ID NO.5, still retains measurable activity against three receptors. This indicates that the peptide system of this invention has modifiability and scalability, and can be further optimized on the basis of peptides to regulate the activity on different receptors. Attached Figure Description

[0025] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used in conjunction with embodiments of the invention to explain the invention and do not constitute a limitation thereof. In the drawings: Figure 1 The results of the RLU preliminary screening experiment of the polypeptide described in this invention in the GLP-1R system; Figure 2 The results of the RLU initial screening experiment of the polypeptide described in this invention in the GCGR system; Figure 3 The results of the RLU initial screening experiment of the polypeptide described in this invention in the GIPR system; Figure 4 The cAMP concentration-reaction curves of the polypeptides and polypeptide derivatives described in this invention in the GLP-1R system are shown. Figure 5 The cAMP concentration-reaction curves of the polypeptides and polypeptide derivatives described in this invention in the GCGR system are shown. Figure 6 The cAMP concentration-reaction curves of the polypeptides and polypeptide derivatives described in this invention in the GIPR system are shown. Figure 7 pEC of the polypeptides and polypeptide derivatives described in this invention in GLP-1R, GCGR and GIPR systems 50 And a comparison chart of 95% confidence intervals. Detailed Implementation

[0026] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings and embodiments. The described embodiments are merely some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0027] Unless otherwise stated, the amino acid raw materials, resins, condensing agents, deprotecting agents and cleavage agents used in the embodiments of the present invention can all be conventional raw materials and reagents in the field of solid-phase peptide synthesis.

[0028] In the following test examples, SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3 correspond to numbers p21, p25 and p31, respectively, and are all C-terminal amidated peptides; SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6 correspond to numbers p21_M, p25_M and p31_M, respectively, and are peptide derivatives obtained by replacing the second amino acid at the N-terminus with Aib and performing esterification modification on a specific lysine side chain based on the corresponding peptide structure, and the peptide derivatives are in the form of C-terminal carboxylic acid.

[0029] Example 1 This embodiment relates to the preparation of peptides, which are prepared using the Fmoc solid-phase peptide synthesis method with Rink Amide-MBHAResin as the solid-phase support to obtain C-terminal amidated products.

[0030] S1. The solid support Rink Amide-MBHA Resin resin was swollen with dichloromethane and then washed with DMF; S2. Remove the Fmoc protecting groups from the resin using an Fmoc deprotection solution, and then sequentially couple the corresponding Fmoc protecting amino acids from the C-terminus to the N-terminus according to the target polypeptide sequence. The Fmoc protecting amino acids include, but are not limited to, Fmoc-His(Trt)-OH, Fmoc-Ala-OH, Fmoc-Ser(tBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Gly-OH, and Fmoc-Thr(tBu)-OH. Fmoc-Phe-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Lys(Boc)-OH, Fm oc-Asn(Trt)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Ile-OH, Fmoc-Trp(Boc)-OH, Fmoc-Leu-OH and Fmoc-Val-OH; S3. After coupling is completed, the coupling reaction can be confirmed by ninhydrin detection, Kaiser test or other conventional solid-phase synthesis detection methods. If the coupling is incomplete, the coupling steps are repeated. S4. After the main chain is assembled, remove the terminal Fmoc protecting group and wash the resin with DMF, dichloromethane and methanol in sequence. S5. Add trifluoroacetic acid lysis buffer to the resin to lyse the target peptide from the resin and remove the side chain protecting groups. After lysis, filter, collect the filtrate, and add cold ether to precipitate. The precipitate is centrifuged, washed with ether, and dried to obtain crude peptide. S6. The crude peptide was purified by reversed-phase high-performance liquid chromatography. Acetonitrile / water system was used as the mobile phase during purification, and gradient elution was set according to the retention behavior of the target peptide. After purification, the main peak component was collected, and after analysis to confirm that the purity was qualified, it was freeze-dried to obtain the target peptide.

[0031] The obtained polypeptide was identified by mass spectrometry, and its molecular weight was consistent with the theoretical molecular weight, with a purity of not less than 95%.

[0032] Example 2 This embodiment relates to the preparation of polypeptide derivatives, which are prepared using the Fmoc solid-phase polypeptide synthesis method.

[0033] S1. For peptide derivatives with a C-terminus of Thr, Fmoc-Thr(tBu)-Wang Resin is used as the solid support. For peptide derivatives with a C-terminus of Gly, Fmoc-Gly-Wang Resin is used as the solid support. When using Wang resin, the product obtained is usually in the form of C-terminal carboxylic acid. S2. Assemble the derivative backbone sequentially from C-terminus to N-terminus according to the target sequence, wherein Fmoc-Aib-OH is coupled at the second position corresponding to the N-terminus of the parent polypeptide, and then Fmoc-His(Trt)-OH is coupled at the N-terminus. S3. After the main chain is assembled, the Fmoc protecting group is removed to obtain the polypeptide main chain containing the His-Aib fragment; S4. Selectively remove the protecting group of the target lysine side chain and couple the AEEA-AEEA-γGlu-C18-diacid modification group to the target lysine side chain. The modification group can be coupled as a pre-synthesized whole fragment, or it can be introduced by gradually coupling AEEA, AEEA, γGlu and C18-diacid. S5. After modification, wash the resin, add lysis buffer to the resin to lyse and remove side chain protecting groups. The lysis product is precipitated with cold diethyl ether, centrifuged, washed and dried to obtain crude peptide. S6. After purification by reversed-phase high-performance liquid chromatography, the main peak component was collected, and after analysis to confirm that the purity was qualified, it was freeze-dried to obtain the target polypeptide derivative.

[0034] In step S2, for lysine residues requiring side-chain esterification modification, it is preferable to use selectively removable side-chain protecting groups, such as Mtt or ivDde; other lysine residues not involved in esterification modification are protected with Boc. Through this protection strategy, selective modification of the target lysine side chain can be achieved.

[0035] The obtained polypeptide derivatives were identified by mass spectrometry, and their molecular weight was consistent with the theoretical molecular weight, with a purity of not less than 95%.

[0036] Test Example 1 RLU screening assay for peptides This test example illustrates the preliminary agonistic activity of the peptide described in this invention on GLP-1R, GCGR, and GIPR.

[0037] (1) Experimental materials Wild-type GLP-1R, GIPR, and GCGR were cloned into the pcDNA3.1 expression vector, and a FLAG tag was inserted after the natural signal peptide. The sequences were confirmed to be correct by sequencing. HEK293FT cells were cultured in DMEM medium containing 10% fetal bovine serum, 50 IU / mL penicillin, and 50 μg / mL streptomycin at 37°C and 5% carbon dioxide.

[0038] (2) Experimental methods Cells were seeded in 6-well plates and cultured overnight. They were then co-transfected with pGloSensor-22F cAMP plasmid and its corresponding receptor plasmid using Lipofectamine 2000. Transfected cells were seeded in 96-well plates at approximately 80,000 cells per well and cultured overnight. D-luciferin was then added to a final concentration of 200 μg / mL, and the plates were incubated at 37°C with 5% CO2 for 1 hour. Afterward, the plates were treated with Hank's Balanced Salt Solution for 10 minutes, followed by the addition of the test peptide dissolved in DMSO. The plates were incubated at room temperature for 20 minutes, and the luciferase signal was detected using a chemiluminescence analyzer. The final concentration of DMSO was consistently maintained at 0.5%. Each group was configured with three replicates.

[0039] (3) Test sample The test peptides were p21, p25, and p31.

[0040] The DMSO group served as a blank control group.

[0041] The Control group served as the positive control group for the corresponding system.

[0042] (4) Experimental results The RLU results of the GLP-1R system are as follows: Figure 1 As shown; RLU results of the GCGR system are as follows Figure 2 As shown; RLU results of the GIPR system are as follows Figure 3 As shown.

[0043] The above results indicate that the three peptides p21, p25, and p31 can all elicit significantly higher luminescent responses than the blank control in the GLP-1R, GCGR, and GIPR systems, suggesting their potential as multi-receptor agonists.

[0044] Test Example 2 cAMP concentration-reaction experiment and pEC of peptides and their derivatives 50 Measurement This test case is used to further confirm the agonistic activity of the polypeptides and their derivatives described in this invention on GLP-1R, GCGR and GIPR, and to compare them with existing reference agonists.

[0045] (1) Experimental methods Cells were collected and resuspended in a detection buffer comprising 5 mM HEPES, 500 μM IBMX, and 0.1% casein. 5 μL of the 2-fold concentration cell suspension was added to a low-volume 384-well plate, approximately 1,000 cells per well for the GLP-1R and GCGR systems, and approximately 2,000 cells per well for the GIPR system. A 10-point gradient dilution of the test peptide was prepared in an independently dilution plate. Then, 5 μL of each concentration of the test sample was added to the detection plate, with replicates for each concentration, and incubated at room temperature for 30 minutes. After incubation, 10 μL of cAMP working assay solution was added, prepared by mixing cAMP lysis buffer, cAMP-D2, and anti-cAMP cryptate reagent in a 38:1:1 volume ratio. After incubation at room temperature in the dark for 1 hour, data were read using an Envision plate reader, and a concentration-response curve was fitted to calculate pEC. 50 Values ​​and 95% confidence intervals.

[0046] (2) Comparison settings In the GLP-1R system, GLP-1(7-37) and retatrutide are used as reference controls; In the GCGR system, glucagon and retatrutide are used as references; In the GIPR system, GIP and retatrutide are used as references.

[0047] (3) Experimental results cAMP concentration-reaction curves of peptides and their derivatives in the GLP-1R system are shown below. Figure 4 As shown; cAMP concentration-reaction curves of peptides and their derivatives in the GCGR system are shown below. Figure 5 As shown; cAMP concentration-reaction curves of peptides and their derivatives in the GIPR system are shown below. Figure 6 As shown.

[0048] corresponding pEC 50 The values ​​and 95% confidence intervals are shown in Table 1.

[0049] Table 1 pEC 50 Table of values ​​and 95% confidence intervals

[0050] As shown in the above charts, in the GLP-1R system, the three peptides p21, p25, and p31 can all induce a significant concentration-dependent cAMP response, and their pEC values ​​are... 50 The p21 pEC values ​​were 11.11, 10.77, and 11.03, respectively, all exhibiting strong GLP-1R agonist activity. Among them, p21 has a pEC value of 11.11, 10.77, and 11.03 on GLP-1R. 50 The p31 pEC value was 11.11, comparable to GLP-1 (7-37) and higher than retatrutide's 10.63; p31 pEC on GLP-1R... 50 The pEC value was 11.03, which is also higher than that of retatrutide. The peptide derivatives p21_M, p25_M, and p31_M also elicited a concentration-dependent response in the GLP-1R system, with pEC values ​​of [missing value]. 50 The values ​​were 9.30, 9.74, and 9.46, respectively, indicating that after Aib replacement and lipolysis modification, GLP-1R agonist activity was still retained, but the overall activity was lower than that of the corresponding peptide.

[0051] In the GCGR system, peptides p21, p25, and p31 all exhibited measurable concentration-dependent cAMP responses, and their pEC values ​​were [missing data]. 50 The values ​​were 9.69, 7.46, and 11.47, respectively. In particular, p31 had a pEC value of 9.69, 7.46, and 11.47 on GCGR. 50 The pEC value reached 11.47, higher than glucagon's 11.26 and retatrutide's 10.69, demonstrating strong GCGR agonist activity. The peptide derivatives p21_M, p25_M, and p31_M also exhibited measurable activity in the GCGR system, with pEC values ​​of [missing value]. 50The values ​​were 7.52, 7.37, and 9.73, respectively, indicating that the GCGR activating ability was still retained after modification, with p31_M retaining relatively high activity.

[0052] In the GIPR system, peptides p21, p25, and p31 can all generate a concentration-dependent cAMP response, and their pEC... 50 The pEC values ​​were 6.39, 6.80, and 7.12, respectively, indicating that all three possess GIPR agonist activity, with p31 exhibiting the highest activity among the three peptides. Regarding peptide derivatives, p25_M still showed measurable activity in the GIPR system, with its pEC value being [missing value]. 50 The value was 6.00; however, p21_M and p31_M did not yield reliable GIPR pEC values ​​under the experimental conditions. 50 value.

[0053] comprehensive Figures 4 to 6 As shown in Table 1, peptides p21, p25, and p31 all possess measurable pEC values ​​in the three receptor systems: GLP-1R, GCGR, and GIPR. 50 The values ​​indicate that all the peptides possess definite triple agonist activity. In contrast, the overall activity profile of the peptide derivatives changed after modification. Among them, p25_M retained measurable triple receptor activity, while p21_M and p31_M mainly retained agonist activity against GLP-1R and GCGR.

[0054] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0055] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. An agonistic peptide targeting GLP-1R / GCGR / GIPR, characterized in that, The amino acid sequences of the polypeptide are shown in SEQ ID NO. 1~3: SEQ ID NO.1: HAQGTFTSDYSKYLDEQAAKEFIAWLVET-NH2; SEQ ID NO.2: HAQGTFTSDYSKYLDKRAQAAFVQWLLNG-NH2; SEQ ID NO. 3: HSQGTFTSDYSKYLDKIAAAEFVQWLLDG-NH2.

2. A polypeptide derivative obtained from the polypeptide of claim 1, characterized in that, The amino acid sequences of the polypeptide derivatives are shown in SEQ ID NO.4~6: SEQ ID NO.4: H-Aib-QGTFTSDYSKYLDEQAAK(AEEA-AEEA-γGlu-C18-diacid)EFIAWLVET-OH; SEQ ID NO.5: H-Aib-QGTFTSDYSKYLDK(AEEA-AEEA-γGlu-C18-diacid)RAQAAFVQWLLNG-OH; SEQ ID NO.6: H-Aib-QGTFTSDYSKYLDK(AEEA-AEEA-γGlu-C18-diacid)IAAAEFVQWLLDG-OH; In this designation, Aib is α-aminoisobutyric acid, AEEA is a hydrophilic linker, γGlu is a γ-glutamyl group, and C18-diacid is a C18 diacid aliphatic chain group.

3. The polypeptide derivative according to claim 2, characterized in that, Aib can be replaced with other non-natural amino acids that can enhance conformational stability, and the AEEA-AEEA-γGlu-C18-diacid esterified side chain can be replaced with other pharmaceutically acceptable aliphatic chain modification groups or linkers.

4. A method for preparing the polypeptide according to claim 1, characterized in that, The Fmoc solid-phase peptide synthesis method was used for preparation, specifically including the following steps: S1. The solid support Rink Amide-MBHA Resin resin was swollen with dichloromethane and then washed with DMF; S2. Remove the Fmoc protecting group on the resin using Fmoc deprotection solution, and then couple the corresponding Fmoc protecting amino acids sequentially from the C-terminus to the N-terminus according to the target polypeptide sequence. S3. After the coupling is completed, check to confirm whether the coupling reaction is complete. If the coupling is incomplete, repeat the coupling steps. S4. After the main chain is assembled, remove the terminal Fmoc protecting group and wash the resin with DMF, dichloromethane and methanol in sequence. S5. Add lysis buffer to the resin to lyse the target peptide from the resin and remove the side chain protecting groups. After lysis, filter the solution, collect the filtrate, and add cold ether to precipitate the peptide. The precipitate is then centrifuged, washed with ether, and dried to obtain the crude peptide. S6. After purification by reversed-phase high-performance liquid chromatography, the main peak component was collected, and after analysis to confirm that the purity was qualified, it was freeze-dried to obtain the target peptide.

5. The method for preparing the polypeptide according to claim 4, characterized in that, The Fmoc-protected amino acids mentioned in step S2 include, but are not limited to, one or more of the following: Fmoc-His(Trt)-OH, Fmoc-Ala-OH, Fmoc-Ser(tBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Gly-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Ile-OH, Fmoc-Trp(Boc)-OH, Fmoc-Leu-OH, and Fmoc-Val-OH; The detection described in step S3 can be performed using conventional solid-phase synthesis detection methods, including but not limited to ninhydrin detection and Kaiser test. The lysis solution described in step S5 is a trifluoroacetic acid system; The purification in step S6 uses an acetonitrile / water system as the mobile phase, and a gradient elution is set according to the retention behavior of the target peptide.

6. A method for preparing the polypeptide derivative according to claim 2, characterized in that, The Fmoc solid-phase peptide synthesis method was used for preparation, specifically including the following steps: S1. Select a solid support based on the C-terminal amino acid of the target polypeptide; S2. Assemble the derivative backbone sequentially from C-terminus to N-terminus according to the target sequence, wherein Fmoc-Aib-OH is coupled at the second position corresponding to the N-terminus of the parent polypeptide, and then Fmoc-His(Trt)-OH is coupled at the N-terminus. S3. After the main chain is assembled, the terminal Fmoc protecting group is removed to obtain the polypeptide main chain containing Aib at the N-terminus. S4. Selectively remove the protecting group of the target lysine side chain and couple the AEEA-AEEA-γGlu-C18-diacid modifying group onto the target lysine side chain; S5. After modification, wash the resin, add lysis buffer to the resin to lyse and remove side chain protecting groups. The lysis product is precipitated with cold diethyl ether, centrifuged, washed and dried to obtain crude peptide. S6. After purification by reversed-phase high-performance liquid chromatography, the main peak component was collected, and after analysis to confirm that the purity was qualified, it was freeze-dried to obtain the target polypeptide derivative.

7. The method for preparing the polypeptide derivative according to claim 6, characterized in that, The selection in step S1 is as follows: when the target peptide C-terminus is Thr, Fmoc-Thr(tBu)-Wang Resin is used as the solid support; when the target peptide C-terminus is Gly, Fmoc-Gly-Wang Resin is used as the solid support. In step S2, for lysine residues that need to be modified by side chain esterification, side chain protecting groups that can be selectively removed, including but not limited to Mtt and ivDde, are used for protection. For lysine residues that do not participate in esterification modification, Boc protecting groups are used for protection. The AEEA-AEEA-γGlu-C18-diacid modifying group mentioned in step S4 can be coupled as a pre-synthesized whole fragment, or it can be introduced by gradually coupling AEEA, AEEA, γGlu and C18-diacid.

8. The use of a polypeptide or polypeptide derivative as an agonist of one or more receptors among GLP-1R, GCGR, and GIPR, characterized in that, The polypeptide is selected from the polypeptide of claim 1, and the polypeptide derivative is selected from the polypeptide derivative of claim 2; The polypeptide has triple agonistic activity against GLP-1R / GCGR / GIPR; the polypeptide derivative has agonistic activity against at least one of the receptors GLP-1R, GIPR, and GCGR.

9. The application according to claim 8, characterized in that, The application is for the preparation of agonist drugs of GLP-1R, GCGR, GIPR or GLP-1R / GCGR / GIPR; The agonist drugs include drugs for the prevention and treatment of diseases with abnormal glucose and lipid metabolism; The disorders of glucose and lipid metabolism include, but are not limited to, obesity, type 2 diabetes, impaired glucose tolerance, insulin resistance, and metabolic syndrome.

10. A pharmaceutical composition, characterized in that, It comprises the polypeptide of claim 1 or the polypeptide derivative of claim 2; The pharmaceutical composition includes an injection solution, a lyophilized powder for injection, a sterile powder for injection, a sustained-release formulation, or a long-acting formulation.

Images