CN114437178B - BIDBH3 mimetic peptide compound targeting PTP1B and its preparation method and application - Google Patents

Abstract

The invention provides a BIDBH3 mimic peptide compound taking PTP1B as a target point, and a preparation method and application thereof, wherein the structure formula of the BIDBH3 mimic peptide compound is shown as follows:

the amino acids in the structure of the BIDBH3 simulated peptide compound are all natural amino acids, and the amino terminal of a peptide chain and R ₁ The radicals being bound by amide bonds, R ₁ Is carboxylic acid or dicarboxylic acid, R ₂ Is OH or NH ₂ . The mimic peptide compound is derived from a core region of a BH3 structural domain of Bcl-2 anti-apoptotic protein and is prepared by a polypeptide solid-phase synthesis method. Experiments prove that the BIDBH3 mimic peptide compound can remarkably inhibit the activity of protein tyrosine phosphorylase 1B (PTP 1B), and has potential application value in the drug development of related diseases taking PTP1B as a target, such as diabetes, cancer, alzheimer disease and the like.

Description

BIDBH3 mimetic peptide compound targeting PTP1B and its preparation method and application

technical field

The invention belongs to the field of biomedicine, and specifically relates to a BIDBH3 mimetic compound targeting PTP1B, a preparation method and application thereof.

Background technique

Protein tyrosine phosphatase 1B (protein tyrosine phosphatase 1B, PTP1B) was successfully isolated and identified as early as 1988. A large number of follow-up research results have shown that the excessive and abnormal expression of PTP1B is closely related to the onset and development of T2DM obesity . PTP1B is a novel potential target for the treatment of T2DM and obesity. PTP1B is a key negative regulatory protein in the insulin signal transduction pathway. PTP1B inhibitors block the insulin-stimulated insulin receptor (IR) tyrosine phosphorylation, thereby affecting the insulin receptor substrate (IRS-1) Phosphorylation, insulin-like and insulin-sensitizing, improving insulin resistance, lowering blood sugar. At the same time, it can enhance the signal of leptin, induce the increase of fat metabolism level, and reduce the body weight. By inhibiting the activity of PTP1B, insulin sensitivity can be enhanced, and insulin resistance in T2DM patients can be effectively improved, and the treatment of T2DM and obesity from the source will be significantly improved. PTP1B is considered to be one of the most ideal targets for the development of non-insulin-dependent anti-type 2 diabetes and obesity drugs. Therefore, finding and developing PTP1B inhibitors with high specificity and low toxicity has very broad application prospects. At present, many pharmaceutical companies such as Pfizer, ISIS, Tabacco, TransTech, etc. have been attracted to develop their specific and high-efficiency inhibitors as new drugs for the treatment of T2DM and obesity. There are also many scientific research institutes in my country (such as Institute of Materia Medica, Chinese Academy of Sciences, Institute of Oceanography, Shanghai Jiaotong University, Zhejiang University, Jilin University, East China University of Science and Technology, Nankai University, Fudan University, China Pharmaceutical University, Sun Yat-sen University, etc.) The basic theory and application research of PTP1B inhibitors make PTP1B inhibitors a very active field in the development of hypoglycemic drugs. A number of candidate compounds, such as Ertiprotafib, TTP814, ISIS-PTPRx, ISIS-113715, MSI-1436, HPN, etc., have successively entered preclinical and clinical phase I and II trials.

Furthermore, recent studies have shown that PTP1B could be a (potential) target for anti-tumor and Alzheimer's drug development. Literature Protein tyrosine phosphatases, new targets for cancer therapy. Curr. Cancer Drug Targets 2006,6,519–532.; A brake becomes anaccelerator: PTP1B-a new therapeutic target for breast cancer. Cancer Cell 2007,11,214–216.; Discover y of [(3 -bromo-7-cyano-2-naphthyl)(difluoro)methyl]-phosphonic acid, a potent and orally active small molecule PTP1B inhibitor. Bioorg. Med. Chem. Lett. 2008, 18, 3200–3205.; Recent advances in the discovery of competitive protein tyrosine phosphatase 1B Inhibitors for the treatment of diabetes, obesity, and cancer. J Med Chem, 53(6) (2010), pp.2333-2344.; PTP1B controls non-mitochondrial oxygen consumption by regulating RNF213 to promote our survival hypoxia. Nat Cell Biol, 18(2016), p.803. found that the overexpression of PTP1B can significantly promote the occurrence and growth of tumors in mice, and the expression of PTP1B through inhibitors can produce anti-tumor effects; the mechanism study found that PTP1B By regulating the RNF213 gene, it controls the non-mitochondrial oxygen consumption of cells, thereby promoting the survival and growth of tumor cells under hypoxic conditions. Accordingly, PTP1B is regarded as a target of antitumor drugs. Literature Protein tyrosine phosphatase 1B (PTP1B): a potential target for Alzheimer'sTherapy? Front Aging Neurosci, 9(7)(2017) summarized the regulatory role of PTP1B in the physiological process related to Alzheimer's disease in the central nervous system in recent years, and proposed that by inhibiting PTP1B and then antagonizing the regulation of PTP1B and Alzheimer's disease The strategy of detrimental physiological processes related to Alzheimer's disease is used for the research and development of anti-Alzheimer's disease drugs.

Therefore, PTP1B has become a popular target for anti-diabetes, cancer and Alzheimer's drug development. The inhibitors of PTP1B that have been discovered so far can be divided into three categories: the first category is inorganic small molecule compounds, represented by sodium vanadate, which have a similar structure to the phosphate radical, the substrate of PTP1B, and can compete with Binds positively to PTP1B and inhibits its activity. However, its selectivity is very low, and it has a strong inhibitory effect on all PTPs, so this type of compound has no prospect of development, and it is impossible to apply it in the middle of clinical treatment. The second category is organic compounds. Most of these substances are screened by organic synthesis and combinatorial chemistry methods. Firstly, compounds with inhibitory PTP1B activity are screened, and then the substituent groups of the compounds are modified to finally obtain a better PTP1B Inhibitors. However, such inhibitors have problems such as poor stability, high charge, and high lipophilic coefficient that restrict the druggability. Although there are already some inhibitors with relatively good selectivity, these PTP1B inhibitors also have an inhibitory effect on TCPTP, which has the highest PTP1B homology. It's a big challenge. In recent years, researchers at home and abroad have set their sights on the third type of PTP1B inhibitors, that is, the research and development of PTP1B inhibitors in natural products. Through high-throughput screening of natural products isolated and identified in nature, some PTP1B inhibitors with high selectivity and activity were obtained although the sites of action were not very clear. Based on the core structure of this natural product, combined with the catalytic active site of PTP1B enzyme, its structure is modified and transformed. In order to develop highly selective, low toxic and highly effective PTP1B inhibitors.

Summarizing and analyzing the development status of PTP1B organic small molecule inhibitors, it can be found that the development of PTP1B small molecule inhibitors suffers from two points: ① The selectivity of PTP1B inhibitors: PTP1B is highly homologous to other protein phosphatases such as TCPTP, especially the activity The homology of the site is as high as 94%; ②PTP1B inhibitor membrane penetration ability: PTP1B catalyzes protein phosphorylation and the active site is charged, so the compounds with PTP1B inhibitory activity are all charged or strongly polar; and PTP1B is distributed in the cell membrane Internally, charged or highly polar compounds have difficulty passing through cell membranes. Therefore, it is very necessary to make up for the defects of existing PTP1B inhibitory molecules and develop new PTP1B inhibitors with novel structures and strong selectivity to meet the urgent needs of domestic clinical practice. The key is to explore and discover new leading structures and modes of action, which is also a hot spot in the current basic research of PTP1B.

Contents of the invention

The object of the present invention is to provide a BIDBH3 mimetic peptide compound targeting PTP1B and its preparation method and application. The BIDBH3 mimetic peptide compound provided by the present invention has significant protein tyrosine phospholipase 1B (PTP1B) inhibitory activity , which can be used for the development of drugs for the prevention or treatment of related diseases with PTP1B as the target.

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

The present invention provides a BIDBH3 mimetic peptide compound targeting PTP1B. The structural formula of the mimetic peptide compound is as follows:

Wherein, R ₁ is carboxylic acid or dicarboxylic acid, R ₂ is OH or NH ₂ .

Further: the amino acids in the BIDBH3 mimetic peptide compound are all natural amino acids, and the amino terminal of the peptide chain is connected to the _R1 group through an amide bond.

Further: The BIDBH3 mimetic peptide compound is specifically:

The present invention also provides the preparation method of the described BIDBH3 mimetic peptide compound, which comprises the following steps:

(1) Resin activation: at room temperature, weigh the corresponding amount of Fmoc-Phe-Wang resin, place it in a manual polypeptide solid-phase synthesizer, and activate it;

(2) adding a mixed solution of piperidine and dimethylformamide to remove the Fmoc protecting group;

(3) Add N-Fmoc protected amino acid, HOBt, HBTU and DIEA in 3 to 4 times the molar amount of the resin, and shake and react at room temperature for 2 to 4 hours;

(4) Repeat steps (2) and (3) until the synthesis of the entire polypeptide sequence is completed;

(5) Drain the resin, add the lysate, shake the shaker, filter, blow nitrogen to remove the remaining trifluoroacetic acid, add ether, centrifuge after the solid is precipitated, and dry to obtain the crude product of the peptidomimetic compound;

(6) The crude product was purified by reversed-phase preparative liquid chromatography, and the mobile phase solution of the target peak was collected to remove acetonitrile, and then freeze-dried to obtain the pure product of the peptidomimetic compound.

Further: the lysate includes phenol, water, thioanisole and trifluoroacetic acid.

The present invention also provides a medicine or a pharmaceutical composition with said BIDBH3 mimetic peptide compound as an active ingredient, comprising any of said BIDBH3 mimetic peptide compounds and one or more pharmaceutically acceptable carriers or excipients.

The present invention also provides the application of the BIDBH3 mimetic peptide compound in the preparation of drugs for preventing or treating diseases with PTP1B as the target.

Further: the diseases include diabetes, cancer and Alzheimer's disease.

Further: the medicine or pharmaceutical composition with the said BIDBH3 mimetic peptide compound as an active ingredient can be administered orally or by injection.

The advantages and technical effects of the present invention are: the present invention provides a BIDBH3 mimetic peptide compound targeting PTP1B and its preparation method and application, and the BIDBH3 mimetic peptide compound can significantly inhibit protein tyrosine phosphorylase 1B (PTP1B) It has potential application value in the development of drugs targeting PTP1B-related diseases such as diabetes, cancer, Alzheimer's disease, etc., and has excellent prospects for the development of PTP1B inhibitors.

Description of drawings

Figure 1 is the PTP1B inhibition rate curve of the mimic peptide compound Pal-PUMA under different concentration gradients;

Figure 2 is the PTP1B inhibition rate curve of the mimic peptide compound Pal-BID under different concentration gradients;

Figure 3 is the PTP1B inhibition rate curve of the mimic peptide compound Pal-BAK under different concentration gradients;

Fig. 4 is the PTP1B inhibition rate curve of the mimetic peptide compound Pal-BIK under different concentration gradients.

Detailed ways:

The technical solutions of the present invention will be further described in detail below in conjunction with specific embodiments.

The BH3 mimetic peptide compounds targeting PTP1B in the present invention are all obtained according to the polypeptide solid-phase synthesis method.

Example 1

1. The specific preparation process of Pal-PUMA is as follows:

(1) Resin activation: at room temperature, weigh the corresponding amount of Fmoc-Phe-Wang resin, wash it with dichloromethane (DCM) 4 times, place it in a manual polypeptide solid-phase synthesizer, add 5ml DCM to swell and activate it for 3 hours, and dimethicone Washed 4 times with methyl formamide (DMF), added 20% piperidine DMF to remove the Fmoc protecting group for 20 min, washed 4 times with 5ml DMF, washed 4 times with 5ml DCM, and tested with Kaiser's reagent.

(2) Connecting Leu(L): DMF was washed 3 times, Fmoc-Leu-OH, HBTU, HOBt and 6 times the resin molar weight of DIEA were added respectively, dissolved in 10ml DMF, stirred at room temperature for 2 hours , washed 4 times with DMF, added 20% piperidine in DMF to remove the Fmoc protecting group for 20 min, washed 4 times with 5ml DMF, 4 times with 5ml DCM, and detected by Kaiser's reagent.

(3) Asp(D) connection: wash with DMF 3 times, add Fmoc-Asp(OtBu)-OH, HBTU, HOBt and 6 times the molar amount of the resin respectively, and dissolve in 10ml DMF, room temperature Stir the reaction for 2 hours, wash with DMF 4 times, add 20% piperidine in DMF to remove the Fmoc protecting group for 20 minutes, wash 4 times with 5ml DMF, wash 4 times with 5ml DCM, and detect with Kaiser's reagent.

(4) Linking Asp(D): DMF was washed 3 times, and Fmoc-Asp(OtBu)-OH, HBTU, HOBt and DIEA of 6 times the molar amount of the resin were added respectively, dissolved in 10ml DMF, and kept at room temperature Stir the reaction for 2 hours, wash with DMF 4 times, add 20% piperidine in DMF to remove the Fmoc protecting group for 20 minutes, wash 4 times with 5ml DMF, wash 4 times with 5ml DCM, and detect with Kaiser's reagent.

(5) Connecting Ala (A): DMF was washed 3 times, respectively added Fmoc-Ala-OH, HBTU, HOBt of 3 times the molar amount of the resin, and DIEA of 6 times the molar amount of the resin, dissolved in 10ml DMF, stirred at room temperature for 2 hours , washed 4 times with DMF, added 20% piperidine in DMF to remove the Fmoc protecting group for 20 min, washed 4 times with 5ml DMF, 4 times with 5ml DCM, and detected by Kaiser's reagent.

(6) Connecting Met(M): DMF was washed 3 times, and Fmoc-Met-OH, HBTU, HOBt and DIEA of 6 times the molar amount of the resin were added respectively, dissolved in 10ml DMF, stirred at room temperature for 2h , washed 4 times with DMF, added 20% piperidine in DMF to remove the Fmoc protecting group for 20 min, washed 4 times with 5ml DMF, 4 times with 5ml DCM, and detected by Kaiser's reagent.

(7) Connecting Arg(R): Washing with DMF for 3 times, adding Fmoc-Arg(Mtr)-OH, HBTU, HOBt and DIEA of 6 times the molar amount of the resin respectively, dissolved in 10ml DMF, and kept at room temperature Stir the reaction for 2 hours, wash with DMF 4 times, add 20% piperidine in DMF to remove the Fmoc protecting group for 20 minutes, wash 4 times with 5ml DMF, wash 4 times with 5ml DCM, and detect with Kaiser's reagent.

(8) Connect Arg(R): DMF washes 3 times, add Fmoc-Arg(Mtr)-OH, HBTU, HOBt and DIEA 6 times the molar amount of the resin respectively, dissolve in 10ml DMF, and keep at room temperature Stir the reaction for 2 hours, wash with DMF 4 times, add 20% piperidine in DMF to remove the Fmoc protecting group for 20 minutes, wash 4 times with 5ml DMF, wash 4 times with 5ml DCM, and detect with Kaiser's reagent.

(9) Connecting Leu(L): wash with DMF for 3 times, add Fmoc-Leu-OH, HBTU, HOBt of 3 times the molar amount of the resin, and DIEA of 6 times the molar amount of the resin respectively, dissolve in 10ml DMF, and stir at room temperature for 2h , washed 4 times with DMF, added 20% piperidine in DMF to remove the Fmoc protecting group for 20 min, washed 4 times with 5ml DMF, 4 times with 5ml DCM, and detected by Kaiser's reagent.

(10) Glu(Q) connection: wash with DMF 3 times, add Fmoc-Glu(OtBu)-OH, HBTU, HOBt and 6 times the molar amount of the resin respectively, and dissolve in 10ml DMF, room temperature Stir the reaction for 2 hours, wash with DMF 4 times, add 20% piperidine in DMF to remove the Fmoc protecting group for 20 minutes, wash 4 times with 5ml DMF, wash 4 times with 5ml DCM, and detect with Kaiser's reagent.

(11) Connecting Ala (A): DMF was washed 3 times, and 3 times the resin molar amount of Fmoc-Ala-OH, HBTU, HOBt and 6 times the resin molar amount of DIEA were added respectively, dissolved in 10ml DMF, stirred at room temperature for 2 hours , washed 4 times with DMF, added 20% piperidine in DMF to remove the Fmoc protecting group for 20 min, washed 4 times with 5ml DMF, 4 times with 5ml DCM, and detected by Kaiser's reagent.

(12) Connecting Gly(G): DMF was washed 3 times, and Fmoc-Gly-OH, HBTU, HOBt and DIEA of 6 times the molar amount of the resin were added respectively, dissolved in 10ml DMF, and stirred at room temperature for 2h , washed 4 times with DMF, added 20% piperidine in DMF to remove the Fmoc protecting group for 20 min, washed 4 times with 5ml DMF, 4 times with 5ml DCM, and detected by Kaiser's reagent.

(13) Connecting Ile (I): DMF washing 3 times, respectively adding 3 times the resin molar amount of Fmoc-Ile-OH, HBTU, HOBt and 6 times the resin molar amount of DIEA, dissolved in 10ml DMF, stirred at room temperature for 2h , washed 4 times with DMF, added 20% piperidine in DMF to remove the Fmoc protecting group for 20 min, washed 4 times with 5ml DMF, 4 times with 5ml DCM, and detected by Kaiser's reagent.

(14) Link palmitic acid (Pal): DMF washes 3 times, add palmitic acid, HBTU, HOBt and DIEA 10 times the molar amount of the resin respectively, dissolve in 10ml DMF, stir at room temperature for 4h, 5ml DMF washed 4 times, 5ml DCM washed 4 times, Kaiser's reagent detection.

(15) Cleavage and solution of side chain protecting group: drain the product, add lysis solution (the lysis solution includes 250mg phenol, 0.5ml water, 0.5ml sulfide anisole and 9.0ml trifluoroacetic acid), stir at room temperature for 2.5h, Filtrate, blow off trifluoroacetic acid with N ₂ , add 30ml of cold anhydrous ether, centrifuge at 5000rpm for 5 minutes to obtain a white precipitate, repeat washing with cold anhydrous ether 3 times, and dry in vacuo to obtain a crude product.

(16) The crude product was purified by reverse-phase preparative liquid chromatography (RP-HPLC), and the target peak was collected. The mobile phase solution was removed from acetonitrile and then freeze-dried to obtain a white solid, that is, the pure product of the BH3 mimetic peptide compound. Through mass spectrometry and high performance liquid chromatography Analysis for structural confirmation.

Example 2

The mass spectrum data and HPLC purity analysis data of the eight BH3 mimetic peptide compounds are shown in Table 1.

Table 1. Mass Spectrometry Data and HPLC Purity Analysis Data of BH3 Mimetic Peptides

Example 3, Determination of protein tyrosine phospholipase 1B (PTP1B) inhibitory activity

In the present invention, MES buffer is adopted as the reaction system, human protein tyrosine phosphatase 1B (PTP1B) is utilized, disodium p-nitrophenylphosphate (pNPP) is used as the specific substrate, and sodium orthovanadate is selected as the positive drug , With DMSO as negative control, a screening model based on enzyme reaction rate of 96-well microplate as carrier was established, and PTP1B inhibitors were found by enzymatic methods.

The specific implementation method is: using MES buffer system (25mM, pH6.5), sequentially add 10μL pNPP (77mM), 86μL MES buffer solution, 4μL compound (2mM compound mother solution dissolved in DMSO), 100μL PTP1B solution ( 50 nM), the total reaction volume was 200 μL. 3 parallels in each group, with DMSO as the negative control and sodium orthovanadate (2mM) as the positive control, at 25°C, shake on the shaker for 1min, read on the microplate reader every 60s, dynamically measure for 5min, and measure the OD 405 change (OD/min). The reaction rate in the initial stage 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 enzyme activity is represented by the rate. The formula for calculating the inhibition rate of the compound on PTP1B:

Inhibition rate (%) = (vDMSO-v sample)/vDMSO*100;

vDMSO and vsample represent the initial average reaction rates of the negative control group and the test compound, respectively.

标示，各组数据运用t检验分析。结果见表2。The obtained data is used for

The data in each group were analyzed using the t test. The results are shown in Table 2.

Table 2 Inhibitory results of tested mimetic peptides on PTP1B activity

In the present invention, the PTP1B inhibition rate and IC ₅₀ of the mimetic peptide compounds Pal-PUMA, Pal-BID, Pal-BAK and Pal-BIK were measured at different concentration gradients, and the results are shown in Table 3 and Figures 1-4.

Table 3 Inhibition rate and IC ₅₀ of polypeptides with different concentration gradients on PTP1B activity

GraphPad Prism 5.0 software was used for statistical processing, and the inhibition rate curve was drawn, as shown in Figure 1 to Figure 4, so as to obtain the PTP1B inhibitory medium concentration IC ₅₀ of the mimic peptide compounds Pal-PUMA, Pal-BID, Pal-BAK and Pal-BIK They were 6.84 μmol/L, 2.15 μmol/L, 1.28 μmol/L, and 0.94 μmol/L, respectively.

The test results showed that the peptidomimetic compounds Pal-PUMA, Pal-BID, Pal-BAK and Pal-BIK showed significant inhibitory effect on protein tyrosine phosphatase 1B, and had excellent prospects for the development of PTP1B inhibitors.

The above embodiments are only used to illustrate the technical solutions of the present invention, rather than limiting them; although the present invention has been described in detail with reference to the foregoing examples, those of ordinary skill in the art can still record the foregoing embodiments Modifications to the technical solutions, or equivalent replacement of some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions claimed in the present invention.

Claims (4)

1. A BIDBH3 mimetic peptide compound targeting PTP1B, characterized in that: the BIDBH3 mimetic peptide compound is specifically:

. 2. The medicine or pharmaceutical composition with the BIDBH3 mimetic peptide compound of claim 1 as an active ingredient, comprising the BIDBH3 mimetic peptide compound and one or more pharmaceutically acceptable carriers or excipients. 3. The use of the BIDBH3 mimetic peptide compound according to claim 1 in the preparation of drugs for the prevention or treatment of diseases targeting PTP1B, characterized in that the diseases are diabetes, cancer and Alzheimer's disease. 4. The application of the BIDBH3 mimetic peptide compound according to claim 3 in the preparation of drugs for the prevention or treatment of diseases targeting PTP1B, characterized in that: the drug with the BIDBH3 mimetic peptide compound as an active ingredient Or the pharmaceutical composition is administered orally or by injection.

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