CN112608375B - Novel BH3 mimetic peptide analogue for inhibiting PTP1B activity and application thereof - Google Patents

Novel BH3 mimetic peptide analogue for inhibiting PTP1B activity and application thereof Download PDF

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CN112608375B
CN112608375B CN202011518318.0A CN202011518318A CN112608375B CN 112608375 B CN112608375 B CN 112608375B CN 202011518318 A CN202011518318 A CN 202011518318A CN 112608375 B CN112608375 B CN 112608375B
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张传亮
苏贤斌
黄鼎旻
王振炜
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Qingdao University of Science and Technology
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Abstract

The invention discloses a novel BH3 mimetic peptide analogue for inhibiting PTP1B activity and application thereof. The structural formula of the novel BH3 mimetic peptide analogs is shown below:
Figure DDA0002848182690000011
the mimic peptide compound is derived from a core region of a Bim-BH3 structural domain, and is prepared by adopting a polypeptide solid-phase synthesis method, and amino acids in the structure are all natural amino acids. The novel BH3 mimetic peptide analogue has remarkable PTP1B inhibition activity, and has potential application value in drug development of related diseases taking PTP1B as a target, such as diabetes, cancer, alzheimer's disease and the like.

Description

Novel BH3 mimetic peptide analogue for inhibiting PTP1B activity and application thereof
Technical Field
The invention belongs to the field of biological medicines, and particularly relates to a novel BH3 mimetic peptide analogue for inhibiting PTP1B activity and application thereof.
Background
Protein tyrosine phosphatase 1B (ptp1b) is closely related to the onset and development of type 2 diabetes and obesity, and is a key negative regulatory protein in the insulin signal transduction pathway. Abnormal overexpression of PTP1B results in decreased insulin sensitivity and development of insulin resistance. The PTP1B inhibitor can block tyrosine phosphorylation of an Insulin Receptor (IR) stimulated by insulin, further influence phosphorylation of an insulin receptor substrate (IRS-1), enhance insulin-like and insulin sensitivity, effectively improve insulin resistance from a source, reduce blood sugar, and simultaneously avoid hypoglycemia adverse reaction of insulin-like medicines. Therefore, PTP1B is also a hot target for the recent study of T2DM, and a number of candidate compounds have been introduced into preclinical and clinical phase I and II experiments. Recent research views in recent years show that: PTP1B can be used as a (potential) target for the development of anti-tumor and Alzheimer's disease drugs. Some researches find that the overexpression of PTP1B can obviously promote the generation and growth of tumors in mice, and the inhibition of the expression of PTP1B by an inhibitor can produce an anti-tumor effect; the mechanism research finds that PTP1B controls non-mitochondrial oxygen consumption of cells by regulating RNF213 gene, and further promotes the survival and growth of tumor cells under the anoxic condition. Thus, PTP1B is considered a target for antitumor drugs. In recent years, PTP1B is also used as a regulation effect in physiological processes related to Alzheimer's disease in the central nervous system, and a strategy for inhibiting PTP1B and further antagonizing harmful physiological processes related to Alzheimer's disease regulated by PTP1B is proposed, so that the development of anti-Alzheimer's disease drugs is carried out. Therefore, PTP1B becomes a potential hot target for development of antidiabetics, cancers and Alzheimer's disease drugs, and the PTP1B inhibitor is expected to be applied to development of antidiabetics, cancers, alzheimer's disease drugs and the like taking PTP1B as a target.
At present, inhibitors of PTP1B mainly comprise small inorganic molecule compounds, organic compounds and PTP1B inhibitors in natural products. But the selectivity of the inorganic micromolecule compound is very low, and the compound has stronger inhibition on all PTPs; most of organic compounds are screened by organic synthesis and combinatorial chemistry methods, a compound with PTP1B activity inhibition is screened firstly, then a substituent group of the compound is modified, and finally a better PTP1B inhibitor is obtained, wherein the inhibitor has the problems of poor stability, higher charge, overhigh lipophilic coefficient and the like which restrict the drug property; PTP1B inhibitors in natural products are obtained by high-throughput screening of natural products isolated and identified in nature, and although they have high selectivity and activity, the site of action is not well-defined. Therefore, it is necessary to make up for the defects of the existing PTP1B inhibitory molecules and develop novel PTP1B inhibitors with novel structures, strong selectivity, low toxicity and high efficiency so as to meet the urgent needs of domestic clinical application.
Disclosure of Invention
The invention provides a novel BH3 mimetic peptide analogue for inhibiting PTP1B activity and application thereof. The novel BH3 mimetic peptide analogue has remarkable PTP1B inhibitory activity and can be used for preparing drugs for preventing or treating related diseases taking PTP1B as a target.
In order to realize the purpose of the invention, the invention adopts the following technical scheme to realize:
the invention provides a novel BH3 mimetic peptide analogue for inhibiting PTP1B activity, which has the following structural formula:
Figure BDA0002848182670000021
wherein R1 is long-chain carboxylic acid, R2 is COOH, and R3 is carboxylic acid or polycarboxylic acid with different chain lengths.
Further, R1 is palmitic acid.
Further, the peptide chain of the BH3 mimetic peptide analogs scan-1-scan-11 adopts an Ala-scanning strategy, and the N-terminal protecting group is modified by palmitic acid. Other such compounds have been modified at the N-terminus of the peptide chain with carboxylic or polycarboxylic acids of varying chain length.
Further, the novel BH3 mimetic peptide analogs are specifically:
Figure BDA0002848182670000031
further, the preparation method of the novel BH3 mimetic peptide analogue comprises the following steps:
(1) Placing Fmoc-Phe-Wang (scan-11 is Fmoc-Ala-Wang) resin in a manual polypeptide solid phase synthesizer at room temperature, and activating by dichloromethane and dimethylformamide;
(2) Adding piperidine/dimethylformamide mixed solution to remove the Fmoc protecting group;
(3) Adding 3-4 times of resin molar weight of N-Fmoc protected amino acid or carboxylic acid, HOBT, HBTU and 5-6 times of resin molar weight of DIEA, and oscillating at room temperature for 2-4 h;
(4) Repeating steps (2) and (3) until the synthesis of the whole mimic peptide sequence is completed;
(5) Adding the lysate into the product obtained in the step (4), stirring at room temperature, filtering, adding anhydrous ether to precipitate a solid, washing, and performing vacuum drying to obtain a crude mimic peptide analogue product;
(6) And purifying the crude peptide analogue product by using a reversed-phase preparative liquid chromatography, collecting a target peak mobile phase solution, removing acetonitrile, and freeze-drying to obtain a flocculent or powdery solid, thus obtaining a pure BH3 mimetic peptide analogue product.
Further, the lysis solution comprises phenol, water, thioanisole and trifluoroacetic acid.
Further, blowing N after filtering in the step (5) 2 Excess trifluoroacetic acid was removed.
The invention also provides a medicament or a pharmaceutical composition which takes the novel BH3 mimetic peptide analogue as an active ingredient, and comprises any novel BH3 mimetic peptide analogue and one or more pharmaceutically acceptable carriers or excipients.
The invention also provides application of the novel BH3 mimetic peptide analogue in preparation of an inhibitor for inhibiting PTP1B activity.
The invention also provides application of the novel BH3 mimetic peptide analogue in preparation of a medicine for preventing or treating diseases taking PTP1B as a target.
Further, the diseases include diabetes, cancer and alzheimer's disease.
Furthermore, the administration mode of the medicine or the pharmaceutical composition taking the novel BH3 mimetic peptide analogue as the active ingredient is oral administration or injection.
Compared with the prior art, the invention has the advantages and the technical effects that:
the invention obtains a novel BimBH3 mimic peptide analogue by a polypeptide solid phase synthesis method, the novel BH3 mimic peptide analogue is derived from a core region of a Bim-BH3 structural domain, and amino acids in the structure are natural amino acids. Experiments prove that the novel BH3 mimetic peptide analogue has a remarkable inhibiting effect on protein tyrosine phosphatase 1B (PTP 1B), and the obtained mimetic peptide analogue has high purity, can be used as an excellent PTP1B inhibitor, and can also be applied to the development of medicaments for related diseases taking PTP1B as a target point, such as diabetes, cancer, alzheimer's disease and the like. Therefore, the novel BH3 mimetic peptide analog has potential application value and good development prospect.
Drawings
FIGS. 1-18 are dose-inhibitory effect curves for the target protein PTP1B for the mimetics scan-2, scan-3, scan-4, scan-5, scan-6, scan-7, scan-8, scan-11, C13-SM6, C14-SM6, C16 diacid-SM6, C18 diacid-SM6, C20-SM6, C22-SM6, C20 diacid-SM6, C22 diacid-SM6, and Lila-SM6, respectively.
Detailed Description
The technical solution of the present invention will be described in further detail with reference to specific examples. The methods in the following examples are conventional methods unless otherwise specified.
Example 1
The specific preparation process of the synthetic route taking scan-1 as an example is as follows:
(1) Resin activation: weighing corresponding amount of Fmoc-Phe-Wang (scan-11 is Fmoc-Ala-Wang) resin, placing the Fmoc-Phe-Wang resin in a manual polypeptide solid phase synthesizer, washing with DCM for 4 times, adding 5ml of DCM for swelling activation for 3h, washing with DMF for 4 times, adding 20% piperidine DMF for removing Fmoc protecting groups twice (20min + 5min), washing with 5ml of DMF for 4 times, washing with 5ml of DCM for 4 times, and detecting with Kaiser's reagent.
(2) Ligation Phe (F): washing with DMF for 3 times, respectively adding Fmoc-Phe-OH, HBTU, HOBt and DIEA with the molar weight 6 times of that of the resin, dissolving in 10ml of DMF, stirring at room temperature for reaction for 2h, washing with DMF for 4 times, adding 20% piperidine DMF to remove Fmoc protecting groups twice (20min + 5min), washing with 5ml of DMF for 4 times, washing with 5ml of DCM for 4 times, and detecting with Kaiser's reagent.
(3) Ligation Glu (E): washing with DMF for 3 times, respectively adding Fmoc-Glu (OtBu) -OH, HBTU, HOBt and DIEA (Diea) with the molar weight 3 times that of the resin, dissolving in 10ml of DMF, stirring at room temperature for reaction for 2h, washing with DMF for 4 times, adding 20% piperidine DMF to remove Fmoc protecting groups twice (20min + 5min), washing with 5ml of DMF for 4 times, washing with 5ml of DCM for 4 times, and detecting with Kaiser's reagent.
(4) Attachment of Asp (D): washing with DMF for 3 times, respectively adding Fmoc-Asp (OtBu) -OH, HBTU, HOBt and DIEA with resin molar amount 3 times that of resin, dissolving in 10ml of DMF, stirring at room temperature for reaction for 2h, washing with DMF for 4 times, adding 20% piperidine DMF to remove Fmoc protecting groups twice (20min + 5min), washing with 5ml of DMF for 4 times, washing with 5ml of DCM for 4 times, and detecting with Kaiser's reagent.
(5) Attachment of Gly (G): washing with DMF for 3 times, respectively adding Fmoc-Gly-OH, HBTU, HOBt and DIEA with the molar weight being 3 times that of the resin, dissolving in 10ml of DMF, stirring at room temperature for reaction for 2h, washing with DMF for 4 times, adding 20% piperidine DMF to remove Fmoc protecting groups twice (20min + 5min), washing with 5ml DMF for 4 times, washing with 5ml DCM for 4 times, and detecting with Kaiser's reagent.
(6) Ligation Ile (I): washing with DMF for 3 times, respectively adding Fmoc-Ile-OH, HBTU, HOBt and DIEA with the molar weight being 3 times that of the resin and 6 times that of the resin, dissolving in 10ml of DMF, stirring at room temperature for reaction for 2h, washing with DMF for 4 times, adding 20% piperidine DMF to remove Fmoc protecting groups twice (20min + 5min), washing with 5ml DMF for 4 times, washing with 5ml DCM for 4 times, and detecting with Kaiser's reagent.
(7) Attachment of Arg (R): washing with DMF for 3 times, respectively adding Fmoc-Arg (Mtr) -OH, HBTU, HOBt and DIEA with resin molar amount 3 times that of the resin, dissolving in 10ml of DMF, stirring at room temperature for reaction for 2h, washing with DMF for 4 times, adding 20% piperidine DMF to remove Fmoc protecting groups twice (20min + 5min), washing with 5ml of DMF for 4 times, washing with 5ml of DCM for 4 times, and detecting with Kaiser's reagent. This step was repeated 1 time.
(8) Attachment of Arg (R): washing with DMF for 3 times, respectively adding Fmoc-Arg (Mtr) -OH, HBTU, HOBt and DIEA with resin molar amount 3 times that of the resin, dissolving in 10ml of DMF, stirring at room temperature for reaction for 2h, washing with DMF for 4 times, adding 20% piperidine DMF to remove Fmoc protecting groups twice (20min + 5min), washing with 5ml of DMF for 4 times, washing with 5ml of DCM for 4 times, and detecting with Kaiser's reagent. This step was repeated 1 time.
(9) Attachment of Leu (L): washing with DMF for 3 times, respectively adding Fmoc-Leu-OH, HBTU, HOBt and DIEA (diethylene glycol ethyl acetate) with the molar weight being 3 times that of the resin, dissolving in 10ml of DMF, stirring at room temperature for reaction for 2h, washing with DMF for 4 times, adding 20% piperidine DMF to remove Fmoc protecting groups twice (20min + 5min), washing with 5ml of DMF for 4 times, washing with 5ml of DCM for 4 times, and detecting with Kaiser's reagent.
(10) Ligation Glu (E): washing with DMF for 3 times, respectively adding Fmoc-Glu (OtBu) -OH, HBTU, HOBt and DIEA (Diea) with the molar weight 3 times that of the resin, dissolving in 10ml of DMF, stirring at room temperature for reaction for 2h, washing with DMF for 4 times, adding 20% piperidine DMF to remove Fmoc protecting groups twice (20min + 5min), washing with 5ml of DMF for 4 times, washing with 5ml of DCM for 4 times, and detecting with Kaiser's reagent.
(11) Link Glu (Q): washing with DMF for 3 times, respectively adding Fmoc-Glu (OtBu) -OH, HBTU, HOBt and DIEA (Diea) with the molar weight 3 times that of the resin, dissolving in 10ml of DMF, stirring at room temperature for reaction for 2h, washing with DMF for 4 times, adding 20% piperidine DMF to remove Fmoc protecting groups twice (20min + 5min), washing with 5ml of DMF for 4 times, washing with 5ml of DCM for 4 times, and detecting with Kaiser's reagent.
(12) Connection to Ala (A): washing with DMF for 3 times, respectively adding Fmoc-Ala-OH, HBTU, HOBt and DIEA with the resin molar weight being 3 times of that of the resin molar weight, dissolving in 10ml of DMF, stirring at room temperature for reaction for 2h, washing with DMF for 4 times, adding 20% piperidine DMF to remove Fmoc protecting groups twice (20min + 5min), washing with 5ml of DMF for 4 times, washing with 5ml of DCM for 4 times, and detecting with Kaiser's reagent.
(13) Connection to Ala (A): washing with DMF for 3 times, respectively adding Fmoc-Ala-OH, HBTU, HOBt and DIEA with resin molar weight being 3 times of that of the resin, dissolving in 10ml of DMF, stirring at room temperature for reaction for 2h, washing with DMF for 4 times, adding 20% piperidine DMF to remove Fmoc protecting groups twice (20min + 5min), washing with 5ml DMF for 4 times, washing with 5ml DCM for 4 times, and detecting with Kaiser's reagent.
(14) Ligation palmitic acid (Pal): washing with DMF for 3 times, respectively adding palmitic acid, HBTU, HOBt and DIEA with the molar weight being 6 times that of the resin and 10 times that of the resin, dissolving in 10ml of DMF, stirring at room temperature for reaction for 4h, washing with 5ml of DMF for 4 times, washing with 5ml of DCM for 4 times, and detecting with Kaiser's reagent.
(15) Cleavage, side chain protecting group cleavage: adding 250mg phenol, 0.5ml water, 0.5ml thioanisole and 9.0ml trifluoroacetic acid into the product, stirring for 2.5h at room temperature, filtering, and adding N 2 Blowing off trifluoroacetic acid, adding 30ml cold anhydrous ether, centrifuging at 5000rpm for 5min to obtain white precipitate, and centrifuging with cold anhydrous etherWashing is repeated for 3 times, and vacuum drying is carried out to obtain a crude product.
(16) Purifying the crude product by reversed phase preparative liquid chromatography (RP-HPLC), collecting a target peak mobile phase solution, removing acetonitrile, freeze-drying to obtain a flocculent or powdery solid, namely a pure BH3 mimetic peptide analogue product, and carrying out structure confirmation by mass spectrometry and high performance liquid chromatography analysis.
Mass spectral data and HPLC purity analytical data for the 27 BH3 mimetic peptide analogs obtained by the above method are shown in table 1.
TABLE 1 Mass Spectrometry data and HPLC purity analysis data for BH3 peptidomimetic analogs
Figure BDA0002848182670000071
Figure BDA0002848182670000081
Example 2: determination of protein tyrosine phospholipase 1B (PTP 1B) inhibitory Activity
According to the invention, MES buffer solution is adopted as a reaction system, human protein tyrosine phosphatase 1B (PTP 1B) is utilized, sodium p-nitrophenylphosphate (pNPP) is adopted as a specific substrate, a lead compound SM-6 is selected as a positive control, DMSO is adopted as a negative control, a screening model with a 96-well microplate based on an enzyme reaction rate as a carrier is established, and a PTP1B inhibitor is searched by an enzymology method.
The specific implementation method comprises the following steps: mu.L of pNPP (77 mM), 86. Mu.L of MES buffer, 4. Mu.L of compound (2 mM), and 100. Mu.L of PTP1B solution (50 nM) were sequentially added to a 96-well plate using MES buffer system (25mM, pH 6.5), and the total reaction volume was 200. Mu.L. Each group of 3 replicates was shaken at 25 ℃ for 1min on a shaker with DMSO as a negative control and sodium orthovanadate (2 mM) as a positive control, read every 60s on a microplate reader, and dynamically measured for 5min for changes in OD 405 (OD/min). The reaction rate in the initial phase of each well is linearly related, and the slope of the linear part of the kinetic curve determines the reaction rate of PTP1B, and the rate indicates the enzyme activity. For the obtained data
Figure BDA0002848182670000082
Showing that each set of data was analyzed using the t-test. The inhibition rate of the compound on PTP1B is calculated by the formula:
inhibition ratio (%) = (v) DMSO -v Sample(s) )/v DMSO ×100％
Wherein v is DMSO 、v Sample(s) The initial average reaction rates of the negative control group and the test compound are expressed respectively
The invention performs PTP1B inhibition rate preliminary screening on the mimic peptide under the concentration of 10 mu mol/L, and performs IC on the compound with the preliminary screening inhibition rate higher than 70 percent 50 The results of the measurements, inhibition, are shown in table 2.
TABLE 2 results of inhibition of PTP1B activity by the tested peptidomimetic analogs
Figure BDA0002848182670000083
Figure BDA0002848182670000091
* : compounds with a preliminary screening inhibition of less than 50% were not IC treated 50 The measurement of (1).
Performing statistical treatment by GraphPad Prism software, drawing an inhibitor quantity effect curve, as shown in figures 1-18, and calculating to obtain PTP1B inhibition medium concentration IC (integrated circuit) of mimic peptide analogs scan-2, scan-3, scan-4, scan-5, scan-6, scan-7, scan-8, scan-11, C13-SM6, C14-SM6, C16 diacid-SM6, C18 diacid-SM6, C20 diacid-SM6, C22 diacid-SM6 and Lila-SM6 50 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, 2875nmol/L, 120.2nmol/L, 384.6nmol/L, 3887nmol/L, 443.5nmol/L, 337.9nmol/L, 199.6nmol/L, 4345nmol/L, respectively.
The test result shows that: the mimic peptide analogue of the invention has obvious inhibition effect on protein tyrosine phosphatase 1B, can be used as an excellent PTP1B inhibitor, and is applied to the development of medicaments for resisting diabetes, tumors and Alzheimer's disease by taking PTP1B as a target, thereby having good development prospect.
The above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof; such modifications and substitutions do not depart from the spirit and scope of the corresponding claims.

Claims (4)

1. A BH3 mimetic peptide analog that inhibits PTP1B activity, said BH3 mimetic peptide analog being specifically:
Figure DEST_PATH_IMAGE002
。
2. a pharmaceutical composition comprising as an active ingredient a BH3 mimetic peptide analog of claim 1, wherein the pharmaceutical composition comprises the BH3 mimetic peptide analog in combination with one or more pharmaceutically acceptable carriers or excipients.
3. Use of the BH3 mimetic peptide analog of claim 1 for the preparation of an inhibitor for inhibiting PTP1B activity.
4. The use of a BH3 mimetic peptide analog of claim 1 in the preparation of a medicament for the prevention or treatment of PTP 1B-targeted diseases, characterized in that: the disease is diabetes, cancer or alzheimer's disease.
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CN202011518318.0A CN112608375B (en) 2020-12-21 2020-12-21 Novel BH3 mimetic peptide analogue for inhibiting PTP1B activity and application thereof
CN202210965783.1A CN115403662A (en) 2020-12-21 2020-12-21 PTP1B polypeptide inhibitor BimBH3-12-Q3A and application thereof
CN202210965782.7A CN115403661A (en) 2020-12-21 2020-12-21 PTP1B polypeptide inhibitor BimBH3-12-G9A and application thereof
CN202210965785.0A CN115403663A (en) 2020-12-21 2020-12-21 PTP1B polypeptide inhibitor BimBH3-12-I8A and application thereof
CN202210965781.2A CN115417922A (en) 2020-12-21 2020-12-21 PTP1B polypeptide inhibitor BimBH3-12-F12A and application thereof
PCT/CN2021/087669 WO2022134395A1 (en) 2020-12-21 2021-04-16 Novel bh3 mimetic peptide analog inhibiting ptp1b activity and application thereof
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CN202210965781.2A Division CN115417922A (en) 2020-12-21 2020-12-21 PTP1B polypeptide inhibitor BimBH3-12-F12A and application thereof
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CN109575108A (en) * 2018-12-12 2019-04-05 中国海洋大学 Using PTP1B as novel B H3 peptide mimics of target spot and its preparation method and application
CN109912688A (en) * 2017-12-12 2019-06-21 青岛海洋生物医药研究院股份有限公司 A kind of PTP1B peptide inhibitor and its preparation method and application
CN110183515A (en) * 2019-04-28 2019-08-30 浙江中医药大学 It is a kind of for PTP1B detection polypeptide and include the fluorescence probe of the polypeptide

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CN115403664A (en) * 2020-12-21 2022-11-29 青岛科技大学 PTP1B polypeptide inhibitor BimBH3-12-R7A and application thereof

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CN109575108A (en) * 2018-12-12 2019-04-05 中国海洋大学 Using PTP1B as novel B H3 peptide mimics of target spot and its preparation method and application
CN110183515A (en) * 2019-04-28 2019-08-30 浙江中医药大学 It is a kind of for PTP1B detection polypeptide and include the fluorescence probe of the polypeptide

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