A PTP1B polypeptide inhibitor BimBH3-12-I8A and its application

Abstract

The present invention discloses a PTP1B peptide inhibitor, BimBH3-12-I8A, having the following structural formula: #imgabs0#. This peptide mimetic compound is derived from the core 12-peptide of the BimBH3 domain, in which Ile (I) at position 8 is replaced by Ala (A). It is prepared using a peptide solid-phase synthesis method, and all amino acids in its structure are natural amino acids. The PTP1B peptide inhibitor, BimBH3-12-I8A, has significant PTP1B inhibitory activity and has potential application in the development of drugs for PTP1B-targeted diseases such as diabetes, cancer, and Alzheimer's disease.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to a PTP1B polypeptide inhibitor BimBH3-12-I8A and applications thereof. Background Art

[0002] Protein tyrosine phosphatase 1B (PTP1B) is closely linked to the pathogenesis and progression of type 2 diabetes and obesity. It is a key negative regulator in the insulin signaling pathway. Abnormal overexpression of PTP1B can reduce insulin sensitivity and lead to insulin resistance. PTP1B inhibitors can block insulin-stimulated tyrosine phosphorylation of the insulin receptor (IR), thereby affecting the phosphorylation of the insulin receptor substrate (IRS-1), enhancing insulin-like production and insulin sensitivity. This effectively addresses insulin resistance at its source, thereby lowering blood sugar levels without the adverse hypoglycemic effects of insulin-based medications. Therefore, PTP1B has become a popular target in recent T2DM research, with several candidate compounds entering preclinical and Phase I / II clinical trials. Recent research suggests that PTP1B may be a potential target for anti-cancer and Alzheimer's disease drug development. Several studies have found that overexpression of PTP1B significantly promotes tumor development and growth in mice, and that inhibiting PTP1B expression with inhibitors can produce anti-tumor effects. Mechanistic studies have revealed that PTP1B regulates the RNF213 gene, thereby controlling non-mitochondrial oxygen consumption and promoting tumor cell survival and growth under hypoxic conditions. Consequently, PTP1B has been identified as a target for anti-tumor drugs. Recently, PTP1B has also been implicated in regulating central nervous system processes associated with Alzheimer's disease. Inhibiting PTP1B has been proposed as a strategy to antagonize the detrimental physiological processes regulated by PTP1B and thus facilitate the development of anti-Alzheimer's drugs. Therefore, PTP1B has become a potential target for the development of anti-diabetes, cancer, and Alzheimer's disease drugs. PTP1B inhibitors are expected to be used in the development of PTP1B-targeted drugs for these conditions.

[0003] Currently, PTP1B inhibitors primarily include inorganic small molecules, organic compounds, and natural product-derived PTP1B inhibitors. However, inorganic small molecules have very low selectivity, exhibiting strong inhibitory activity against all PTPs. Organic compounds are typically screened through organic synthesis and combinatorial chemistry, initially identifying compounds with PTP1B inhibitory activity and then modifying their substituents to ultimately yield a promising PTP1B inhibitor. However, these inhibitors suffer from poor stability, high charge, and excessive lipophilicity, hindering their drugability. Natural product-derived PTP1B inhibitors are typically screened through high-throughput screening of naturally isolated and identified natural products. While these inhibitors exhibit high selectivity and activity, their sites of action are not well defined. Therefore, it is imperative to address the limitations of existing PTP1B inhibitors and develop novel, structurally novel, highly selective, low-toxic, and highly effective PTP1B inhibitors to meet the urgent clinical needs in China. Summary of the Invention

[0004] The present invention provides a PTP1B polypeptide inhibitor BimBH3-12-I8A and its application. The PTP1B polypeptide inhibitor BimBH3-12-I8A has significant PTP1B inhibitory activity and can be used to develop drugs for preventing or treating diseases targeting PTP1B.

[0005] In order to achieve the above-mentioned purpose, the present invention adopts the following technical solutions:

[0006] The present invention provides a novel BH3 mimetic peptide analog that inhibits PTP1B activity. The structural formula of the novel BH3 mimetic peptide analog is as follows:

[0007]

[0008] Among them, R1 is a long-chain carboxylic acid, R2 is COOH, and R3 is a carboxylic acid or polycarboxylic acid with different carbon chain lengths.

[0009] The present invention provides a PTP1B polypeptide inhibitor BimBH3-12-I8A, the structural formula of the PTP1B polypeptide inhibitor BimBH3-12-I8A is as follows:

[0010]

[0011] Furthermore, the preparation method of the PTP1B polypeptide inhibitor BimBH3-12-I8A comprises the following steps:

[0012] (1) Place Fmoc-Phe-Wang resin in a manual peptide solid phase synthesizer at room temperature and activate with dichloromethane and dimethylformamide;

[0013] (2) adding a piperidine / dimethylformamide mixture to remove the Fmoc protecting group;

[0014] (3) Add 3-4 times the molar amount of the resin to the N-Fmoc protected amino acid or carboxylic acid, HOBT, HBTU, and 5-6 times the molar amount of the resin to the DIEA, and shake the reaction at room temperature for 2-4 hours;

[0015] (4) repeating steps (2) and (3) until the synthesis of the entire peptide mimetic sequence is completed;

[0016] (5) adding the lysate to the product of step (4), stirring at room temperature, filtering, adding anhydrous ether to precipitate the solid, washing, and vacuum drying to obtain a crude product of the peptidomimetic analog;

[0017] (6) The crude peptide analog product is purified by reverse phase preparative liquid chromatography, and the target peak mobile phase solution is collected and acetonitrile is removed, and then freeze-dried to obtain a flocculent or powdery solid, thereby obtaining a pure product of the PTP1B polypeptide inhibitor BimBH3-12-I8A.

[0018] Furthermore, the lysis solution includes phenol, water, thioanisole and trifluoroacetic acid.

[0019] Furthermore, in step (5), N2 is blown after filtration to remove excess trifluoroacetic acid.

[0020] The present invention also provides a medicine or pharmaceutical composition with the PTP1B polypeptide inhibitor BimBH3-12-I8A as an active ingredient, comprising any of the PTP1B polypeptide inhibitors BimBH3-12-I8A and one or more pharmaceutically acceptable carriers or excipients.

[0021] The present invention also provides the use of the PTP1B polypeptide inhibitor BimBH3-12-I8A in the preparation of an inhibitor for inhibiting PTP1B activity.

[0022] The present invention also provides the use of the PTP1B polypeptide inhibitor BimBH3-12-I8A in the preparation of a drug for preventing or treating diseases targeting PTP1B.

[0023] Furthermore, the diseases include diabetes, cancer and Alzheimer's disease.

[0024] Furthermore, the drug or pharmaceutical composition containing the PTP1B polypeptide inhibitor BimBH3-12-I8A as an active ingredient is administered orally or by injection.

[0025] Compared with existing technologies, the present invention offers the following advantages and technical effects: A PTP1B peptide inhibitor, BimBH3-12-I8A, is obtained through solid-phase peptide synthesis. This peptide mimetic compound is derived from the core 12-peptide of the BimBH3 domain, in which Ile at position 8 is replaced by Ala. All amino acids in its structure are naturally occurring amino acids. This novel BH3 mimetic peptide analog exhibits significant PTP1B inhibitory activity and has potential application in the development of drugs targeting PTP1B-related diseases, such as diabetes, cancer, and Alzheimer's disease. Therefore, the PTP1B peptide inhibitor, BimBH3-12-I8A, possesses potential application value and promising development prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The dose-inhibitory effect curve of the PTP1B polypeptide inhibitor molecule BimBH3-12-I8A (i.e., scan-7) on the target protein PTP1B. DETAILED DESCRIPTION

[0027] The technical solutions of the present invention are further described in detail below with reference to specific embodiments. The methods in the following embodiments are all conventional methods unless otherwise specified.

[0028] Example 1

[0029] Taking scan-1 as an example, the specific preparation process is as follows:

[0030] (1) Resin activation: Weigh the corresponding amount of Fmoc-Phe-Wang (Fmoc-A1a-Wang for scan-11) resin and place it in a manual peptide solid phase synthesizer. Wash it with DCM four times, add 5 ml of DCM to swell and activate for 3 h, wash it with DMF four times, add 20% piperidine in DMF to remove the Fmoc protecting group twice (20 min + 5 min), wash it with 5 ml of DMF four times, wash it with 5 ml of DCM four times, and detect it with Kaiser's reagent.

[0031] (2) Attachment of Phe(F): Wash with DMF three times, add 3 times the molar amount of resin Fmoc-Phe-OH, HBTU, HOBt and 6 times the molar amount of resin DIEA, respectively, dissolve in 10 ml DMF, stir at room temperature for 2 h, wash with DMF four times, add 20% piperidine in DMF to remove the Fmoc protecting group twice (20 min + 5 min), wash with 5 ml DMF four times, wash with 5 ml DCM four times, and detect with Kaiser's reagent.

[0032] (3) Glu(E) attachment: Wash with DMF three times, add 3 times the molar amount of resin Fmoc-Glu(OtBu)-OH, HBTU, HOBt and 6 times the molar amount of resin DIEA, dissolve in 10 ml DMF, stir at room temperature for 2 h, wash with DMF four times, add 20% piperidine in DMF to remove the Fmoc protecting group twice (20 min + 5 min), wash with 5 ml DMF four times, wash with 5 ml DCM four times, and detect with Kaiser's reagent.

[0033] (4) Attachment of Asp(D): Wash with DMF three times, add 3 times the molar amount of resin, Fmoc-Asp(OtBu)-OH, HBTU, HOBt and 6 times the molar amount of resin, dissolve in 10 ml of DMF, stir at room temperature for 2 h, wash with DMF four times, add 20% piperidine in DMF to remove the Fmoc protecting group twice (20 min + 5 min), wash with 5 ml of DMF four times, wash with 5 ml of DCM four times, and detect with Kaiser's reagent.

[0034] (5) Attachment of Gly(G): Wash with DMF three times, add 3 times the molar amount of resin Fmoc-Gly-OH, HBTU, HOBt and 6 times the molar amount of resin DIEA, dissolve in 10 ml DMF, stir at room temperature for 2 h, wash with DMF four times, add 20% piperidine in DMF to remove the Fmoc protecting group twice (20 min + 5 min), wash with 5 ml DMF four times, wash with 5 ml DCM four times, and detect with Kaiser's reagent.

[0035] (6) Connecting Ile(I): Wash with DMF three times, add 3 times the molar amount of resin Fmoc-Ile-OH, HBTU, HOBt and 6 times the molar amount of resin DIEA, dissolve in 10 ml DMF, stir at room temperature for 2 h, wash with DMF four times, add 20% piperidine in DMF to remove the Fmoc protecting group twice (20 min + 5 min), wash with 5 ml DMF four times, wash with 5 ml DCM four times, and detect with Kaiser's reagent.

[0036] (7) Arg(R) attachment: Wash with DMF three times. Add 3 times the molar amount of the resin, Fmoc-Arg(Mtr)-OH, HBTU, HOBt, and 6 times the molar amount of the resin, respectively, and dissolve in 10 ml of DMF. Stir and react at room temperature for 2 h. Wash with DMF four times. Add 20% piperidine in DMF to remove the Fmoc protecting group twice (20 min + 5 min). Wash with 5 ml of DMF four times and 5 ml of DCM four times. Detect with Kaiser's reagent. Repeat this step once.

[0037] (8) Arg(R) attachment: Wash with DMF three times. Add 3 times the molar amount of the resin, Fmoc-Arg(Mtr)-OH, HBTU, HOBt, and 6 times the molar amount of the resin, respectively, and dissolve in 10 ml of DMF. Stir and react at room temperature for 2 h. Wash with DMF four times. Add 20% piperidine in DMF to remove the Fmoc protecting group twice (20 min + 5 min). Wash with 5 ml of DMF four times and 5 ml of DCM four times. Detect with Kaiser's reagent. Repeat this step once.

[0038] (9) Attachment of Leu (L): Wash with DMF three times, add 3 times the molar amount of resin Fmoc-Leu-OH, HBTU, HOBt and 6 times the molar amount of resin DIEA, dissolve in 10 ml DMF, stir at room temperature for 2 h, wash with DMF four times, add 20% piperidine in DMF to remove the Fmoc protecting group twice (20 min + 5 min), wash with 5 ml DMF four times, wash with 5 ml DCM four times, and detect with Kaiser's reagent.

[0039] (10) Glu(E) attachment: Wash with DMF three times, add 3 times the molar amount of resin Fmoc-Glu(OtBu)-OH, HBTU, HOBt and 6 times the molar amount of resin DIEA, dissolve in 10 ml DMF, stir at room temperature for 2 h, wash with DMF four times, add 20% piperidine in DMF to remove Fmoc protecting group twice (20 min + 5 min), wash with 5 ml DMF four times, wash with 5 ml DCM four times, and detect with Kaiser's reagent.

[0040] (11) Glu(Q) attachment: Wash with DMF three times, add 3 times the molar amount of resin Fmoc-Glu(OtBu)-OH, HBTU, HOBt and 6 times the molar amount of resin DIEA, dissolve in 10 ml DMF, stir at room temperature for 2 h, wash with DMF four times, add 20% piperidine in DMF to remove Fmoc protecting group twice (20 min + 5 min), wash with 5 ml DMF four times, wash with 5 ml DCM four times, and detect with Kaiser's reagent.

[0041] (12) Attachment of Ala(A): Wash with DMF three times, add 3 times the molar amount of resin Fmoc-Ala-OH, HBTU, HOBt and 6 times the molar amount of resin DIEA, dissolve in 10 ml DMF, stir at room temperature for 2 h, wash with DMF four times, add 20% piperidine in DMF to remove the Fmoc protecting group twice (20 min + 5 min), wash with 5 ml DMF four times, wash with 5 ml DCM four times, and detect with Kaiser's reagent.

[0042] (13) Attachment of Ala(A): Wash with DMF three times, add 3 times the molar amount of resin Fmoc-Ala-OH, HBTU, HOBt and 6 times the molar amount of resin DIEA, dissolve in 10 ml DMF, stir at room temperature for 2 h, wash with DMF four times, add 20% piperidine in DMF to remove the Fmoc protecting group twice (20 min + 5 min), wash with 5 ml DMF four times, wash with 5 ml DCM four times, and detect with Kaiser's reagent.

[0043] (14) Attachment of palmitic acid (Pal): Wash with DMF three times, add 6 times the molar amount of palmitic acid, HBTU, HOBt and 10 times the molar amount of DIEA, dissolve in 10 ml of DMF, stir at room temperature for 4 h, wash with 5 ml of DMF four times, wash with 5 ml of DCM four times, and detect with Kaiser's reagent.

[0044] (15) Cleavage and removal of side chain protecting groups: 250 mg of phenol, 0.5 ml of water, 0.5 ml of thioanisole, and 9.0 ml of trifluoroacetic acid were added to the product, stirred at room temperature for 2.5 h, filtered, and the trifluoroacetic acid was blown off with N2. 30 ml of cold anhydrous ether was added, and the mixture was centrifuged at 5000 rpm for 5 min to obtain a white precipitate. The precipitate was washed three times with cold anhydrous ether and dried in vacuo to obtain a crude product.

[0045] (16) The crude product was purified by reverse phase preparative liquid chromatography (RP-HPLC), and the target peak mobile phase solution was collected to remove acetonitrile, and then freeze-dried to obtain a flocculent or powdery solid, i.e., the pure BH3 mimetic peptide analog. The structure was confirmed by mass spectrometry and high performance liquid chromatography analysis.

[0046] The mass spectrometry data and HPLC purity analysis data of 27 BH3 mimetic peptide analogs obtained by the above method are shown in Table 1.

[0047] Table 1 Mass spectrometry data and HPLC purity analysis data of BH3 mimetic peptide analogs

[0048]

[0049] Example 2: Protein tyrosine phospholipase 1B (PTP1B) inhibitory activity assay

[0050] In the present invention, MES buffer was used as the reaction system, human protein tyrosine phosphatase 1B (PTP1B) was utilized, disodium p-nitrophenylphosphate (pNPP) was used as the specific substrate, the lead compound SM-6 was selected as the positive control, and DMSO was used as the negative control. A 96-well microplate screening model based on enzyme reaction rate was established to search for PTP1B inhibitors through enzymatic methods.

[0051] The specific implementation method is as follows: using MES buffer system (25mM, pH6.5), 10μL pNPP (77mM), 86μL MES buffer, 4μL compound (2mM), 100μL PTP1B solution (50nM) were added sequentially in a 96-well plate, and the total reaction volume was 200μL. Each group was paralleled 3 times, with DMSO as the negative control and sodium orthovanadate (2mM) as the positive control. The reaction was shaken on a shaker at 25°C for 1min, and the microplate reader was read every 60s for 5min. The kinetic measurement was performed to measure the change in OD 405 (OD / min). The reaction rate in the initial stage of each well was linearly correlated. The slope of the linear part of the kinetic curve determined the reaction rate of PTP1B, and the enzyme activity was expressed as speed. The data obtained were used The data of each group were analyzed by t-test. The calculation formula of the inhibition rate of compound on PTP1B is:

[0052] Inhibition rate (%) = (v DMSO -v 样本 ) / v DMSO ×100%

[0053] Among them, v DMSO 、v 样本 Represent the initial average reaction rates of the negative control group and the test compound, respectively

[0054] The present invention performs a preliminary screening of the PTP1B inhibition rate of the mimetic peptide at a concentration of 10 μmol / L, and performs IC 50 The inhibition results are shown in Table 2.

[0055] Table 2 Inhibition results of the tested peptide analogs on PTP1B activity

[0056]

[0057]

[0058] * : Compounds with inhibition rates below 50% in the initial screening were not subjected to IC 50 Determination of.

[0059] GraphPad Prism software was used for statistical analysis to draw the inhibitory dose-effect curve (see Figure 1). The PTP1B inhibition median concentration IC of the mimetic peptide analogs scan-2, scan-3, scan-4, scan-5, scan-6, scan-7, scan-8, scan-11, C13-SM6, C14-SM6, C16diacid-SM6, C18-SM6, C18diacid-SM6, C20-SM6, C20diacid-SM6, C22-SM6, C22diacid-SM6, and Lila-SM6 were calculated. 50 They are 91.6nmol / L, 703.0nmol / L, 580.9nmol / L, 1208.0nmol / L, 56.5nmol / L, 45.4nmol / L, 63.7nmol / L, 511.9nmol / L, 835.4nmol / L, 262.7nmol / L, respectively. 2875nmol / L, 120.2nmol / L, 384.6nmol / L, 3887nmol / L, 443.5nmol / L, 337.9nmol / L, 199.6nmol / L, 4345nmol / L.

[0060] The experimental results show that the peptidomimetic analogs of the present invention exhibit significant inhibitory effects on protein tyrosine phosphatase 1B, can be used as excellent PTP1B inhibitors, and can be used in the development of anti-diabetic, anti-tumor and anti-Alzheimer's disease drugs targeting PTP1B, and therefore have good development prospects.

[0061] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for a person skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to replace some of the technical features therein with equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions claimed to be protected by the present invention.

Claims

1. A PTP1B polypeptide inhibitor BimBH3-12-I8A, characterized in that The structural formula of the PTP1B polypeptide inhibitor BimBH3-12-I8A is as follows: 。 2. A drug or pharmaceutical composition comprising the PTP1B polypeptide inhibitor BimBH3-12-I8A according to claim 1 as an active ingredient, characterized in that: The medicine or pharmaceutical composition comprises the PTP1B polypeptide inhibitor BimBH3-12-I8A and one or more pharmaceutically acceptable carriers or excipients.

3. Use of the PTP1B polypeptide inhibitor BimBH3-12-I8A according to claim 1 in the preparation of a drug for preventing or treating a disease targeting PTP1B, characterized in that: The diseases targeting PTP1B are diabetes and Alzheimer's disease.

4. The use according to claim 3, characterized in that: The drug or pharmaceutical composition containing the PTP1B polypeptide inhibitor BimBH3-12-I8A as an active ingredient is administered orally or by injection.

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