A class of hapten and fatty acid double modified bim bh3 mimic peptide, and preparation method and application thereof

Abstract

This invention discloses a novel class of long-acting hypoglycemic BimBH3 mimic peptides, modified with both a hapten and fatty acid targeting PTP1B, along with their preparation methods and applications. The general structural formula of the BimBH3 mimic peptide is shown in Formula I. These compounds are derived from the core region of the Bim-BH3 domain. The second amino acid is replaced by a non-natural amino acid modified with a hapten to increase the peptide's stability against DPP-4 enzyme. A long-chain fatty acid (hexadecanoic acid or octadecanoic acid) is conjugated at the N-terminus to ensure good PTP1B inhibitory activity, resulting in a BimBH3 mimic peptide with both longer-lasting and higher activity, exerting its hypoglycemic effect by inhibiting PTP1B. All the compounds described above were prepared using a solid-phase peptide synthesis method with 2-CTC resin. The crude products were cut, purified, and lyophilized to obtain the pure target compounds.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a class of long-acting hypoglycemic BimBH3 mimic peptides that target PTP1B hapten and fatty acid dual modification, as well as their preparation method and application. Background Technology

[0002] Recent studies have shown that protein tyrosine phosphatase 1B (PTP1B) is a key negative regulator of the insulin signaling pathway, closely related not only to the pathogenesis and development of type 2 diabetes and obesity, but also as a potential target for anti-tumor and Alzheimer's disease drug development. Traditional small-molecule PTP1B inhibitors often suffer from poor selectivity, poor cell membrane permeability, and insufficient efficacy in lowering blood sugar and reducing weight in vivo. For example, candidate molecules such as ertiprotafib, MSI-1436, and JTT-551, which entered clinical trials, were ultimately terminated due to poor efficacy or adverse reactions, and are considered undruggable targets. Therefore, drugs targeting PTP1B for diabetes, obesity, cancer, and Alzheimer's disease remain to be developed.

[0003] BH3 peptides are key fragments of the BH3 domain of Bcl-2 family pro-apoptotic proteins and are crucial molecules regulating mitochondrial apoptosis signaling pathways. In recent years, international research has shifted towards the various physiological functions of Bcl-2 family proteins beyond apoptosis regulation, such as metabolic regulation and mitochondrial morphology regulation. Recent studies have shown that the BH3-only pro-apoptotic protein Bad and its BH3 mimic peptides participate in the regulation of mitochondrial nutrition and energy metabolism. For example, the phosphorylated BadBH3 and BadBH3 mimics modified using a Hydrocarbon-Stapled strategy, reported by Nika Danial's group at Harvard University, can restore glucose-driven mitochondrial respiration, the function and number of damaged pancreatic β-cells, and insulin secretion by targeting glucokinase, exhibiting potential anti-T2DM activity. (Nat.Struct.Mol.Biol.,2013,21:36.;Cell Rep.,2015,10(4):497-504.) Inspired by this, the applicant's previous research identified six BH3 mimic peptide molecules that can effectively bind to PTP1B and inhibit its activity from more than 10 Bcl-2 pro-apoptotic protein BH3 domain peptides. Further, fatty acid acylation was used for long-acting modification, and a series of BimBH3 peptide-derived PPT1B / TC-PTP inhibitors were discovered. Their potential for improving insulin resistance in vitro / in vivo and for long-acting anti-T2DM once a week, as well as their weight loss benefits, were confirmed. (ACS Med.Chem.Lett.,2021,12(6):1017-23.;J.Med.Chem.2023,66,(4),3030–3044).

[0004] The development of peptide drugs faces two main challenges: first, peptides are generally unstable and degrade rapidly in the body; second, most bioactive peptides have poor bioavailability and cannot be taken orally, necessitating the development of suitable routes of administration through dosage form modifications. Endogenous antibodies are naturally occurring antibodies abundant in the systemic circulation, such as 2,4-dinitrophenyl (DNP) and 1,3-diketone (DK). DNP haptens act as endogenous antibody binders, and natural anti-DNP antibodies are abundant in the blood of humans of all sexes, races, and ages. Using aptamers composed of targeting small molecules linked to the corresponding hapten, peptide hapten conjugates and antibody complexes can be successfully formed in vivo, effectively masking active peptide molecules, reducing degradation by proteases, and prolonging their half-life. For example, Masanobu Nagano's team demonstrated that reactive immunization using a KLH-bound 1,3-diketone (DK) hybrid can generate aldolase-catalyzing antibodies. Wu Zhimeng's team designed the GLP-1 analog exinum 4 (Ex4) as a model peptide to bind with a pre-existing endogenous antibody, forming a complex. In pharmacokinetic (PK) and hypoglycemic activity studies, compared with the natural Ex4 antibody, the hapten conjugate Ex4-DNP showed significantly improved half-life and long-lasting antidiabetic activity (J.Med.Chem.2021,64,4947-4959). This mechanism, similar to a peptide-Fc fusion strategy, prolongs the peptide's half-life and greatly avoids potential immunogenicity issues. This rationally designed peptide-hapten conjugate can bind with endogenous antibodies to form a peptide-hapten-antibody complex, maximizing the extension of the peptide drug's half-life while retaining its hypoglycemic efficacy.

[0005] Injectable peptide hypoglycemic drugs often require frequent injections, leading to poor patient adherence. This is one of the main reasons why about half of type 2 diabetes mellitus (T2DM) patients have unsatisfactory glycemic control. Therefore, once-weekly long-acting T2DM treatments that significantly improve the convenience, adherence, and quality of life for T2DM patients are increasingly favored by patients and the market. Currently, various modification strategies can extend the half-life of peptide / protein drugs, enabling once-weekly or even once-every-two-week dosing. These include PEGylation, glycosylation, amino acid sequence modification, introduction of non-natural amino acids, fatty acid modification, human serum albumin fusion, Fc fusion, and the development of sustained-release delivery systems and oral formulations. Among these, fatty acid acylation technology was proposed early and is widely used. Popular weekly formulations like semaglutide and telpolide in recent years utilize fatty acid acylation technology. Fatty acid side chains are typically long-chain fatty acids with 12-22 carbons, which can reversibly bind to albumin in the body, effectively reducing the metabolism and clearance of fatty acid-acylated peptide-like compounds and significantly extending the drug's half-life. Furthermore, this technology can alter the distribution of peptides and may have the potential for tissue targeting. Due to the inherent safety and well-defined chemical properties of fatty acids, this technology has become an important method for the discovery of long-acting peptide and protein drugs. (Nat Rev Drug Discov, 22(1), 59–80.)

[0006] The BH3 mimic peptide derivatives described in this invention are derived from the core region of the BimBH3 domain. Simultaneously, employing a fatty acid acylation and hapten modification strategy, the N-terminus of the peptide chain is conjugated with a fatty acid capable of reversibly binding to serum albumin. The second amino acid is modified using a non-natural amino acid derived from the hapten 2,4-dinitrophenyl (DNP), which can bind to specific endogenous antibodies, further prolonging the half-life of the mimic peptide. Theoretically, this can significantly extend the duration of the hypoglycemic effect, achieving a long-acting hypoglycemic effect with once-weekly dosing. In summary, this type of BimBH3 mimic peptide, modified with both hapten and fatty acid, is a long-acting peptide hypoglycemic drug targeting PTP1B and possessing the potential for long-term once-weekly hypoglycemic action. Summary of the Invention

[0007] This invention provides a class of long-acting hypoglycemic BimBH3 mimic peptides that target PTP1B with dual modification of a hapten and fatty acids, as well as their preparation method and applications. The BimBH3 mimic peptide analogs exhibit good PTP1B enzyme inhibitory activity, with the preferred compound Y5 showing excellent DPP-4 enzyme stability. Furthermore, in type 2 diabetic db / db mice, they demonstrate excellent once-weekly long-acting hypoglycemic effect and weight loss benefits, and can be used for the development of long-acting drugs to prevent or treat PTP1B-targeted diseases (preferably type 2 diabetes and obesity).

[0008] To achieve the above-mentioned objectives, the present invention employs the following technical solution:

[0009] This invention discloses a novel class of long-acting hypoglycemic BimBH3 mimic peptide compounds that are dual-modified with a PTP1B hapten and fatty acids, as well as their preparation method and applications. The general structural formula of the BimBH3 mimic peptide is shown in Formula I:

[0011] in,

[0013] These compounds are derived from the core peptide region of the Bim-BH3 domain. A fatty acid (hexadecanoic acid or octadecanoic acid) capable of reversibly binding to serum albumin is conjugated to the N-terminus of the peptide chain. The second amino acid is modified using a non-natural amino acid derived from the hapten 2,4-dinitrophenyl (DNP), which can bind to specific endogenous antibodies, further prolonging the half-life of the mimic peptide. Theoretically, this can significantly prolong the duration of the hypoglycemic effect, resulting in a longer-acting and more active BimBH3 mimic peptide that exerts its hypoglycemic effect by inhibiting PTP1B. All the compounds described above were prepared using a solid-phase peptide synthesis method with 2-CTC resin. The crude products were cut, purified, and lyophilized to obtain the pure target compounds.

[0014] Furthermore, the preparation method of the novel BimBH3 mimic peptide includes the following steps:

[0015] (1) At room temperature, 2-CTC resin was placed in a glass peptide solid-phase synthesizer and swollen with DCM for 30 min;

[0016] (2) Add 3 times the molar amount of N-Fmoc to protect phenylalanine and 3.6 times the molar amount of DIEA to the resin, and react with nitrogen bubbling at room temperature for 60 min;

[0017] (3) Add piperidine / DMF mixture to remove Fmoc protecting groups;

[0018] (4) Add 2 times the resin molar amount of N-Fmoc to protect amino acids and 2.4 times the resin molar amount of HOBt and DIC, and react under nitrogen bubbling at 40℃ for 30-60 min;

[0019] (5) Repeat steps (3) and (4) until the synthesis of the first ten amino acid sequences is completed;

[0020] (6) Add piperidine / DMF mixture to remove Fmoc protecting groups;

[0021] (7) Add 4 times the resin molar amount of N-Fmoc to protect the amino acid modified by the hapten and 4.8 times the resin molar amount of HOBt, HBTU and DIEA, and react with nitrogen bubbling at 40℃ for 60 min.

[0022] (8) Add piperidine / DMF mixture to remove Fmoc protecting groups;

[0023] (9) Add 4 times the resin molar amount of N-Fmoc protected isoleucine and 4.8 times the resin molar amount of HOBt and DIC, and react under nitrogen bubbling at 40°C for 30-60 min.

[0024] (10) Add piperidine / DMF mixture to remove Fmoc protecting groups;

[0025] (11) Add 4 times the resin molar amount of hexadecanoic acid or octadecanoic acid and 4.8 times the resin molar amount of HOBt, HBTU and DIEA, and react under nitrogen bubbling at 40℃ for 60-120 min.

[0026] (12) Wash 3 times with DMF, 3 times with DCM, and 3 times with MeOH, then dry.

[0027] (13) Add lysis buffer to the product of step (12), shake at 40°C for 2 hours, filter, add anhydrous ice ether to precipitate solid, wash and vacuum dry to obtain crude product of simulated peptide analog.

[0028] (14) The crude peptide analog product is purified by reversed-phase preparative liquid chromatography. After the target peak is collected and the mobile phase solution is deacetonitrile, it is freeze-dried to obtain flocculent or powdered solid, which is the pure Bim BH3 mimic peptide modified with hapten.

[0029] Furthermore, the pyrolysis solution comprises triisopropylsilane, 3,6-dioxa-1,8-octanedithiol, water, and trifluoroacetic acid.

[0030] Furthermore, in step (13), excess trifluoroacetic acid is removed by blowing N2 after filtration.

[0031] The present invention also provides a drug or pharmaceutical composition having as an active ingredient a novel long-acting hypoglycemic BimBH3 mimic peptide that targets PTP1B and is dual-modified with fatty acids, as described in any one of claims 1-5, characterized in that the drug or pharmaceutical composition comprises any one of the novel long-acting hypoglycemic BimBH3 mimic peptides that targets PTP1B and is dual-modified with fatty acids and one or more pharmaceutically acceptable carriers or excipients.

[0032] Furthermore, the novel long-acting hypoglycemic BimBH3 mimic peptide, which is dual-modified with a hapten and fatty acids targeting PTP1B, is used to prepare an inhibitor that inhibits PTP1B activity.

[0033] Furthermore, the novel long-acting hypoglycemic BimBH3 mimic peptide, which is dual-modified with a hapten and fatty acids targeting PTP1B, is used to prepare drugs for the prevention or treatment of diseases targeting PTP1B.

[0034] Furthermore, the diseases include diabetes, cancer, and Alzheimer's disease, with preferred indications being type 2 diabetes and obesity (once-weekly long-acting treatment);

[0035] Furthermore, the drug or pharmaceutical composition with the novel long-acting hypoglycemic BimBH3 mimic peptide, which is dual-modified with PTP1B hapten and fatty acid, as the active ingredient is administered via subcutaneous injection or oral administration.

[0036] Compared with existing technologies, the advantages and beneficial effects of this invention are as follows: This invention obtains a novel long-acting hypoglycemic BimBH3 mimic peptide with dual modification of PTP1B hapten and fatty acid through a peptide solid-phase synthesis method. This type of compound is derived from the core peptide region of the Bim-BH3 domain, and adopts a fatty acid acylation and hapten modification strategy. The N-terminus of the peptide chain is conjugated with a fatty acid (hexadecanoic acid or octadecanoic acid) that can reversibly bind to serum albumin. The second amino acid is modified with a non-natural amino acid derived from the hapten 2,4-dinitrophenyl (DNP), which can bind to specific endogenous antibodies to further prolong the half-life of the mimic peptide. Compared with currently available long-acting hypoglycemic drugs, it has a novel target, a significantly simpler structure (12 amino acids), and a significantly lower preparation cost than once-weekly GLP-1 receptor agonists such as semaglutide (39 amino acids) and telposide (39 amino acids). This invention experimentally demonstrates that the BimBH3 mimic peptide exhibits a significant inhibitory effect on protein tyrosine phosphatase 1B (PTP1B), and the obtained mimic peptide has high purity, making it an excellent PTP1B inhibitor for drug development targeting PTP1B-related diseases such as diabetes, obesity, cancer, and Alzheimer's disease. Of particular note is that this invention, through activity screening, yielded a BimBH3 mimic peptide with significantly improved stability against DPP-4, long-acting in vivo glucose-lowering effects, and weight-loss benefits. Therefore, the BimBH3 analogue described in this invention has significant development value and excellent commercial prospects in the field of long-acting glucose-lowering drugs targeting PTP1B. Attached Figure Description

[0037] Figure 1 Stability of BimBH3 naked peptide and mimetic peptide Y5 to DPP-4 enzyme.

[0038] Figure 2 Effects of mimic peptide Y5 on protein expression levels in insulin-resistant HepG2 cells.

[0039] Figure 3 The peptide Y5 mimics long-lasting hypoglycemic activity and weight loss benefits in vivo. Detailed Implementation

[0040] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. Unless otherwise specified, the methods in the following embodiments are conventional methods.

[0041] Example 1

[0042] The synthetic route for compound Y1 is as follows, and the specific preparation process is as follows:

[0043] (1) Resin activation: Weigh the appropriate amount of 2-CTC resin and place it in a polypeptide solid-phase synthesizer. Add 5 ml of DCM to swell and activate for 1 h.

[0044] (2) Phe(F) ligation: Wash 3 times with DMF, add 3 times the molar amount of Fmoc-Phe-OH and 3.6 times the molar amount of DIEA to 10 ml of DCM, and bubble at room temperature for 1 h, then wash 4 times with DMF. Block the resin 3 times with 5 ml of blocking buffer (DCM:MeOH:DIEA = 17:2:1), 10 min each time, then wash 4 times with DMF. Add 20% piperidine DMF to remove the Fmoc protecting group twice (8 min + 15 min), wash 4 times with 5 ml of DMF, and detect with Kaiser's reagent.

[0045] (3) Glu(E) linkage: Wash 3 times with DMF, add 2 times the resin molar amount of Fmoc-Glu(OtBu)-OH, 2.4 times the resin molar amount of HOBt and DIC respectively, dissolve in 10 ml DMF, bubble reaction at 40℃ for 30-60 min, wash 4 times with DMF, add 20% piperidine DMF to remove Fmoc protecting group twice (8 min + 15 min), wash 4 times with 5 ml DMF, and detect with Kaiser's reagent.

[0046] (4) Asp(D) ligation: Wash 3 times with DMF, add 2 times the resin molar amount of Fmoc-Asp(OtBu)-OH, 2.4 times the resin molar amount of HOBt and DIC respectively, dissolve in 10 ml DMF, bubble reaction at 40℃ for 30-60 min, wash 4 times with DMF, add 20% piperidine DMF to remove Fmoc protecting group twice (8 min + 15 min), wash 4 times with 5 ml DMF, and detect with Kaiser's reagent.

[0047] (5) Connecting Gly(G): Wash 3 times with DMF, add 2 times the resin molar amount of Fmoc-Gly-OH, 2.4 times the resin molar amount of HOBt and DIC respectively, dissolve in 10 ml DMF, bubble reaction at 40℃ for 30-60 min, wash 4 times with DMF, add 20% piperidine DMF to remove Fmoc protecting group twice (8 min + 15 min), wash 4 times with 5 ml DMF, and detect with Kaiser's reagent.

[0048] (6) Ile(I) ligation: Wash 3 times with DMF, add 2 times the resin molar amount of Fmoc-Ile-OH, 2.4 times the resin molar amount of HOBt and DIC respectively, dissolve in 10 ml DMF, bubble reaction at 40℃ for 30-60 min, wash 4 times with DMF, add 20% piperidine DMF to remove Fmoc protecting group twice (8 min + 15 min), wash 4 times with 5 ml DMF, and detect with Kaiser's reagent.

[0049] (7) Connecting Arg(R): Wash 3 times with DMF, add 2 times the resin molar amount of Fmoc-Arg(Mtr)-OH, 2.4 times the resin molar amount of HOBt and DIC respectively, dissolve in 10 ml DMF, bubble reaction at 40℃ for 30-60 min, wash 4 times with DMF, add 20% piperidine DMF to remove Fmoc protecting group twice (8 min + 15 min), wash 4 times with 5 ml DMF, and detect with Kaiser's reagent.

[0050] (8) Connecting Arg(R): Wash 3 times with DMF, add 2 times the resin molar amount of Fmoc-Arg(Mtr)-OH, 2.4 times the resin molar amount of HOBt and DIC respectively, dissolve in 10 ml DMF, bubble reaction at 40℃ for 30-60 min, wash 4 times with DMF, add 20% piperidine DMF to remove Fmoc protecting group twice (8 min + 15 min), wash 4 times with 5 ml DMF, and detect with Kaiser's reagent.

[0051] (9) Ligation of Leu(L): Wash 3 times with DMF, add 2 times the resin molar amount of Fmoc-Leu-OH, 2.4 times the resin molar amount of HBTU, HOBt and DIEA respectively, dissolve in 10 ml DMF, bubble reaction at 40℃ for 30-60 min, wash 4 times with DMF, add 20% piperidine DMF to remove Fmoc protecting group twice (8 min + 15 min), wash 4 times with 5 ml DMF, and detect with Kaiser's reagent.

[0052] (10) Glu(E) linkage: Wash 3 times with DMF, add 2 times the resin molar amount of Fmoc-Glu(OtBu)-OH, 2.4 times the resin molar amount of HOBt and DIC respectively, dissolve in 10 ml DMF, bubble reaction at 40℃ for 30-60 min, wash 4 times with DMF, add 20% piperidine DMF to remove Fmoc protecting group twice (8 min + 15 min), wash 4 times with 5 ml DMF, and detect with Kaiser's reagent.

[0053] (11) Connecting Gln(Q): Wash 3 times with DMF, add 2 times the resin molar amount of Fmoc-Gln(OtBu)-OH, 2.4 times the resin molar amount of HOBt and DIC respectively, dissolve in 10 ml DMF, bubble reaction at 40℃ for 30-60 min, wash 4 times with DMF, add 20% piperidine DMF to remove Fmoc protecting group twice (8 min + 15 min), wash 4 times with 5 ml DMF, and detect with Kaiser's reagent.

[0054] (12) Ligation of Lys(DNP): Wash 3 times with DMF, add 4 times the resin molar amount of Fmoc-Lys(DNP)-OH, 4.8 times the resin molar amount of HBTU, HOBt and DIEA respectively, dissolve in 10 ml DMF, bubble reaction at 40℃ for 60 min, wash 4 times with DMF, add 20% piperidine DMF to remove Fmoc protecting group twice (8 min + 15 min), wash 4 times with 5 ml DMF, and detect with Kaiser's reagent.

[0055] (13) Ile(I) ligation: Wash 3 times with DMF, add 4 times the resin molar amount of Fmoc-Ile-OH, 4.8 times the resin molar amount of HBTU, HOBt and DIEA respectively, dissolve in 10 ml DMF, bubble reaction at 40℃ for 30-60 min, wash 4 times with DMF, add 20% piperidine DMF to remove Fmoc protecting group twice (8 min + 15 min), wash 4 times with 5 ml DMF, and detect with Kaiser's reagent.

[0056] (14) Connecting hexadecanoic acid: Wash 3 times with DMF, add 4 times the resin molar amount of palmitic acid, 4.8 times the resin molar amount of HBTU, HOBt and DIEA respectively, dissolve in 10 ml DMF, bubble reaction at 40℃ for 1 h, wash 4 times with 5 ml DMF, and detect with Kaiser's reagent.

[0057] (15) Wash 3 times with 5ml DCM, wash 3 times with 5ml DMF, wash 3 times with 5ml MeOH, and then dry.

[0058] (16) Cleavage and splitting of side chain protecting groups: Add 0.25 ml triisopropylsilane, 0.25 ml water, 0.25 ml 3,6-dioxa-1,8-octanedithiol and 9.25 ml trifluoroacetic acid to the product, shake at 40 °C for 2 h, filter, blow off the trifluoroacetic acid with N2, add 30 ml anhydrous ice ether, centrifuge at 3000 rpm for 3 min to obtain a white precipitate, wash three times with cold anhydrous ether, and vacuum dry to obtain the crude product.

[0059] (17) The crude product was purified by reversed-phase preparative liquid chromatography (RP-HPLC), and the target peak was collected. After removing acetonitrile from the mobile phase solution, it was freeze-dried to obtain a flocculent or powdered solid, namely the pure BimBH3 mimic peptide modified with hapten. The structure was confirmed by mass spectrometry and high-performance liquid chromatography.

[0060] The mass spectrometry data and HPLC purity analysis data of the 25 BimBH3 mimic peptide compounds described in this invention are shown in Table 1.

[0061] Table 1. Mass spectrometry data and HPLC purity analysis data of the hapten-modified BH3 mimic peptide.

[0063] Example 2: Determination of protein tyrosine phospholipase 1B (PTP1B) inhibitory activity

[0064] In this invention, MES buffer was used as the reaction system, human protein tyrosine phosphatase 1B (PTP1B) was used, p-nitrophenyl phosphate disodium (pNPP) was used as the specific substrate, SM-6 was selected as the lead compound as the positive control and DMSO was used as the negative control, and a screening model based on enzyme reaction rate was established using 96-well microplates as the carrier to search for PTP1B inhibitors through enzymatic methods.

[0065] The specific implementation method is as follows: Using a MES buffer system (25mM, pH 6.5), 10μL of pNPP (77mM), 86μL of MES buffer, 4μL of the compound (2mM), and 100μL of PTP1B solution (50nM) were added sequentially to a 96-well plate, for a total reaction volume of 200μL. Each group was divided into three replicates, with DMSO as a negative control and sodium orthovanadate (2mM) as a positive control. The plate was shaken on a shaker at 25℃ for 1 min, and the OD values ​​were read at 1 min and 5 min using a microplate reader. 405 Value, calculate OD 405 The rate of change V (ΔOD / min). The initial reaction rate of each well is linearly correlated, and the slope of the linear portion of the kinetic curve determines the reaction rate of PTP1B, with rate representing enzyme activity. The obtained data are used...

[0066] Inhibition rate (%) = (V DMSO -v 样本 ) / V DMSO ×100%

[0067] Among them, v DMSO v 样本 The initial mean response rates of the negative control group and the test compound are represented, respectively. Statistical analysis and processing of inhibition rate data were performed using GraphPad Prism software, and inhibitor-response curves were fitted and IC50 values ​​were calculated. 50 value.

[0068] Table 2. Inhibition results of the tested peptide analogues on PTP1B activity

[0070] The experimental results show that the peptide analogues described in this invention exhibit significant inhibitory effects on protein tyrosine phosphatase 1B, and can be used as excellent PTP1B inhibitors in the development of anti-diabetic, anti-tumor, and anti-Alzheimer's disease drugs targeting PTP1B.

[0071] Example 3: Stability study of mimic peptide Y5 against DPP-4

[0072] (1) Preparation of simulated peptide Y5 sample: Take out the lyophilized peptide sample powder from the -80℃ freezer, weigh 1 mg of Y5 peptide sample, and dissolve it in 1 mL of sodium phosphate buffer (pH = 7.0, 20 mM) to make a peptide sample solution with a concentration of 1 mg / mL. Take out the DPP-4 enzyme (≥45 U / mg) from -80℃ and thaw it slowly. Accurately measure 4.5 μL of the thawed DPP-4 enzyme with a pipette and add it to the peptide sample solution and mix well.

[0073] (2) Simulated peptide Y5 and DPP-4 enzyme reaction and sampling analysis: The reaction system was incubated in a 37℃ water bath, and samples were taken at different time points: 0 min, 5 min, 15 min, 30 min, 60 min, and 120 min. At each time point, 100 μL of the reaction solution was taken using a pipette, and 200 μL of sodium bicarbonate (0.1 M) was added to terminate the reaction. The quenched sample was vortexed for 30 s, centrifuged at 14000 r / min for 10 min. Finally, the supernatant was collected, avoiding the bottom precipitate, and filtered through a 0.22 μm sterile filter for sterilization. 30 μL of each supernatant was then taken for HPLC analysis.

[0074] (3) Data processing: Based on the HPLC integral area of ​​each simulated peptide sample, the relative number (%) of remaining peptides at each time point was calculated. Half-life was calculated by plotting the percentage of remaining peptides against reaction time, fitting the data to the exponential decay model in GraphPad Prism 5 software, and comparing it with the half-life of BimBH3-SM6.

[0075] Experimental results are as follows Figure 1 As shown, the second position in the BimBH312 peptide sequence is Ala, which is theoretically a specific degradation site for DPP-4 and can be rapidly metabolized and degraded by it. Figure 1 As shown in Figure A, the unmodified BimBH312 peptide degrades rapidly with a half-life of approximately 18 min. In contrast, the mimic peptide Y5 exhibits stronger anti-metabolic stability against DPP-4, with a half-life as long as 13.78 h, approximately 45 times that of the naked peptide. Figure 1 B). These results indicate whether N-terminal fatty acylation and site-specific substitution of amino acids at the 2-position using hapten modification can improve the antimetabolite stability of the mimic peptide Y5 against DPP-4.

[0076] Example 4: Evaluation of the effect of mimicking peptide Y5 on improving insulin resistance at the cellular level

[0077] (1) Cellular insulin resistance model: HepG2 cells in the logarithmic growth phase were prepared into a single-cell suspension using DMED medium containing 10% FBS and seeded at 3000 cells per well in 96-well plates. The cells were divided into CTL group, Model group and drug treatment group. After the cells were attached to the plate in monolayer, the CTL group was treated with INS only, the Model group was treated with INS and PA, and the drug treatment group was treated with INS, PA and mimic peptide Y5 (5 μM and 10 μM). The cells were incubated for 24 hours. Before the end of the incubation, the cells were treated with INS for 10 min.

[0078] (2) Western blotting was used to detect changes in the expression levels of signal molecules.

[0079] Preparation of SDS-PAGE gel: Mount clean electrophoresis glass plates onto the gel casting rack as required, and add an appropriate amount of double-distilled water to check for leaks. Add an appropriate amount of water, 30% polyacrylamide, and separating gel buffer or stacking gel buffer to the centrifuge tube according to the required gel concentration. After adding the coagulant APS and TEMED, mix immediately. Use a pipette to add the mixed gel solution between the mounted glass plates. The separating gel volume should be approximately 5 mL, and the stacking gel volume approximately 3 mL. After adding the stacking gel, immediately insert the 15-well comb that fits the glass plates, taking care to avoid air bubbles.

[0080] Sample collection: When collecting adherent cells, discard the culture medium and wash with PBS at low temperature 2-3 times. Finally, aspirate any remaining PBS, add cell lysis buffer, and place on ice for 15 minutes to completely lyse the cells. Collect the cells, centrifuge at 12,000 rpm for 15 minutes at 4°C, and collect the supernatant. Detect the protein content in the supernatant using a BCA protein assay kit. First, equilibrate the protein content with cell lysis buffer, then add an equal volume of 2× loading buffer and mix well. Finally, transfer to a metal bath and boil at 95°C for 10 minutes.

[0081] Electrophoresis: Add 15 μg of total protein to each well of the SDS-PAGE gel. The electrophoresis conditions for the upper gel are 90 V for 30 min. The electrophoresis conditions for the lower gel are 120 V for 90 min. Stop electrophoresis when the bromophenol blue in the buffer reaches the bottom of the gel plate.

[0082] Transfer: Wet transfer was performed after electrophoresis. Prepare 1× transfer buffer (20% ethanol): 10× transfer buffer, double-distilled water, and anhydrous ethanol. Activate the PVDF membrane beforehand, assembling it in the following order: positive electrode - filter paper - PVDF membrane - gel - filter paper - negative electrode, taking care to avoid air bubbles. Transfer conditions: 200mA current, 90min. Blocking: Immerse the transferred PVDF membrane in the prepared 5% skim milk powder. Block slowly on a shaker at room temperature for 1 hour. After blocking, wash 3-5 times and cut out the band containing the target protein according to the marker position. Incubation with primary antibody: Dilute the primary antibody in advance using 5% skim milk powder prepared by TBST. The bands incubated with the primary antibody can be directly incubated on a shaker at room temperature for 1 hour or overnight at 4°C.

[0083] Secondary antibody incubation: Recover the primary antibody, thoroughly wash the PVDF membrane with TBST to remove unbound primary antibody, and incubate on a shaker at room temperature for 8 minutes each time. Dilute the corresponding secondary antibody and incubate on a shaker at room temperature for 1 hour.

[0084] Development: Discard the secondary antibody, wash the PVDF membrane 5 times with TBST, and shake at room temperature for 8 minutes each time. Prepare the chemiluminescence solution 10 minutes before development according to the instructions. Detect using a chemiluminescence imaging system.

[0085] Experimental results are as follows Figure 2 As shown. PTP1B is a negative regulator of the insulin signaling pathway, and the mimic peptide Y5 improves insulin resistance by inhibiting PTP1B activity and activating IR-β and Akt. Figure 2 As shown, compared with the control group, after treatment of insulin-resistant model cells at concentrations of 5 and 10 μM for 24 h, the expression level of PTP1B by the mimic peptide Y5 was not reduced, but the phosphorylation levels of IR-β and Akt were significantly increased. This indicates that the mimic peptide Y5 significantly improved the insulin resistance induced by PA in HepG2 cells.

[0086] Example 5: Evaluation of the long-lasting hypoglycemic activity and weight loss benefits of the highly active PTP1B inhibitor BimBH3 mimic peptide compound in vivo

[0087] Evaluation of in vivo hypoglycemic effect: Female C57BL / Ks db / db mice, 8-9 weeks old, were normally housed in an animal room with a constant temperature of 24±2.0℃ and a normal day-day cycle. After one week of acclimatization feeding, fasting blood glucose was measured. Mice with blood glucose levels exceeding 11 mM were randomly divided into three groups: a parallel control group (physiological saline), a positive control group (semaglutide 0.1 μmol / kg), and a treatment group (mimicking peptide compound Y5: 0.5 μmol / kg, 0.1 μmol / kg, and 0.05 μmol / kg). Five mice were assigned to each group, for a total of five groups. The medication was administered subcutaneously (10 mL / 10 g mouse body weight) once a week for 7 weeks. Blood glucose levels were measured by tail blood sampling using an ACCU-CHEK Performa blood glucose meter (Roche Diabetes Care Gmbh Sandhofer Strasse 11668305 Mannheim, Germany). Changes in blood glucose levels in type 2 diabetic mice in each group were monitored, and mouse body weight was recorded during the treatment period.

[0088] The in vivo long-acting hypoglycemic activity results of the highly active PTP1B inhibitor BimBH3 mimic peptide compound described in this invention are shown in the figure. Figure 3 As shown. Fasting blood glucose levels were measured in each group on day 1 before the experiment. The medication was administered once on days 1, 8, 15, 22, 29, 36, and 43. Figure 3 The experimental results of A, 3B, and 3C show that the fasting blood glucose level of the model mice in the parallel control group remained elevated, which is consistent with the pathological characteristics of type 2 diabetes. However, the fasting blood glucose level of the mice in the treatment groups with different doses was slightly lower than that of the parallel control group at 1-2 weeks to significantly lower than that of the parallel control group at 6-7 weeks. This indicates that the BimBH3 mimic peptide compound (Y5) with excellent PTP1B inhibitory activity described in this invention exhibits excellent long-acting anti-type 2 diabetes therapeutic effects.

[0089] Furthermore, the weight loss effect evaluation of the highly active PTP1B inhibitor BimBH3 mimic peptide compound described in this invention is shown in [link to relevant documentation]. Figure 1 As shown in D. Weight was measured in each group on day 1, and the medication was administered once on days 1, 8, 15, 22, 29, 36, and 43. Figure 3 The experimental results of D show that the average weight gain of mice in the treatment group at different doses was lower than that of mice in the parallel control group. This indicates that the BimBH3 mimic peptide compound (Y5) with excellent PTP1B inhibitory activity described in this invention not only exhibits excellent long-term hypoglycemic activity in vivo, but also shows certain weight loss benefits and therapeutic potential for anti-obesity.

[0090] In summary, this invention provides a class of long-acting hypoglycemic BimBH3 mimic peptides that target PTP1B with dual modification of a hapten and fatty acids, along with their preparation method and applications. The BimBH3 mimic peptide analogs exhibit good PTP1B enzyme inhibitory activity, with the preferred compound Y5 showing excellent DPP-4 enzyme stability. Furthermore, in type 2 diabetic db / db mice, they demonstrate excellent once-weekly long-acting hypoglycemic effect and weight loss benefits. These analogs can be used for the development of long-acting drugs to prevent or treat PTP1B-targeted diseases (preferably type 2 diabetes and obesity), showing high development potential and excellent application value.

[0091] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions claimed by the present invention.

Claims

1. A novel long-acting hypoglycemic BimBH3 mimic peptide that targets PTP1B with both hapten and fatty acid modification, wherein the general structural formula of the BimBH3 mimic peptide is shown in Formula I: ； Formula I in, ； The N-terminal R1 is modified with hexadecanoic acid or octadecanoic acid, and the R2 is a non-natural amino acid modified with a hapten. This not only retains its high PTP1B enzyme inhibitory activity, but also, after the introduction of hapten modification, it can bind to specific endogenous antibodies to further prolong the half-life of the mimic peptide.

2. The method for preparing the novel long-acting hypoglycemic BimBH3 mimic peptide with dual modification of PTP1B hapten and fatty acid as described in claim 1, characterized in that, The preparation method includes the following steps: (1) At room temperature, 2-CTC resin was placed in a glass peptide solid-phase synthesizer and swollen with DCM for 30 min; (2) Add 3 times the molar amount of N-Fmoc to protect phenylalanine and 3.6 times the molar amount of DIEA to the resin, and react under nitrogen bubbling at room temperature for 60 min; (3) Add piperidine / DMF mixture to remove Fmoc protecting groups; (4) Add 2 times the resin molar amount of N-Fmoc to protect amino acids and 2.4 times the resin molar amount of HOBt and DIC, and react under nitrogen bubbling at 40℃ for 30~60min; (5) Repeat steps (3) and (4) until the synthesis of the first ten amino acid sequences is complete; (6) Add piperidine / DMF mixture to remove Fmoc protecting groups; (7) Add 4 times the resin molar amount of N-Fmoc to protect the amino acid modified by the hapten and 4.8 times the resin molar amount of HOBt, HBTU and DIEA, and react with nitrogen at 40℃ for 60 min. (8) Add piperidine / DMF mixture to remove Fmoc protecting groups; (9) Add 4 times the molar amount of N-Fmoc-protected isoleucine and 4.8 times the molar amount of HOBt and DIC, and react under nitrogen bubbling at 40°C for 30~60 min. (10) Add piperidine / DMF mixture to remove Fmoc protecting groups; (11) Add 4 times the molar amount of hexadecanoic acid or octadecanoic acid and 4.8 times the molar amount of HOBt, HBTU and DIEA to the resin, and react under nitrogen bubbling at 40℃ for 60-120 min. (12) Wash three times with DMF, three times with DCM, and three times with MeOH; (13) Add lysis buffer to the product of step (12), shake at 40°C for 2 hours, filter, add anhydrous ice ether to precipitate solid, wash and vacuum dry to obtain crude product of simulated peptide analog; (14) The crude peptide analog product is purified by reversed-phase preparative liquid chromatography. After the target peak is collected and the mobile phase solution is deacetonated, it is freeze-dried to obtain a flocculent or powdered solid, which is the pure Bim BH3 hapten-modified analog peptide.

3. The preparation method according to claim 2, characterized in that, The pyrolysis solution described in the preparation step includes triisopropylsilane, 3,6-dioxa-1,8-octanedithiol, water, and trifluoroacetic acid.

4. The preparation method according to claim 2, characterized in that, In step (13), after filtration, N2 is blown to remove excess trifluoroacetic acid.

5. A drug or pharmaceutical composition having as its active ingredient the novel long-acting hypoglycemic BimBH3 mimic peptide, which is a dual-modified PTP1B hapten and fatty acid according to claim 1, or the novel long-acting hypoglycemic BimBH3 mimic peptide prepared by the preparation method according to any one of claims 3-4, characterized in that, The drug or pharmaceutical composition comprises any of the novel PTP1B-targeting hapten and fatty acid-modified long-acting hypoglycemic BimBH3 mimic peptides and one or more pharmaceutically acceptable carriers or excipients.

6. The use of the novel long-acting hypoglycemic BimBH3 mimic peptide, which is a dual-modified PTP1B hapten and fatty acid according to claim 1, or the novel long-acting hypoglycemic BimBH3 mimic peptide prepared by the preparation method according to any one of claims 3-4, in the preparation of an inhibitor for inhibiting PTP1B activity, characterized in that... The inhibitor is used to treat diabetes and obesity.

7. The use of the novel long-acting hypoglycemic BimBH3 mimic peptide, which is a dual-modified PTP1B hapten and fatty acid according to claim 1, or the novel long-acting hypoglycemic BimBH3 mimic peptide prepared by the preparation method according to any one of claims 3-4, in the preparation of a medicament for the prevention or treatment of diseases targeting PTP1B, characterized in that, The diseases mentioned are diabetes and obesity.

8. The application according to claim 7, characterized in that, The drug in question is a long-acting medication for the treatment of diabetes and obesity.

9. The application according to claim 7, characterized in that, The drug or drug composition with the mimetic peptide as the active ingredient is administered orally or by injection.

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