A polypeptide for inhibiting the expression of glutamine transporter II, a preparation method thereof, and applications thereof

Abstract

This application relates to the field of biological medicine technology, and specifically discloses a polypeptide for inhibiting the expression of glutamine transporter II, its preparation method and application. The polypeptide has (i) an amino acid sequence as shown in SEQ ID No. 1; or (ii) an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence as described in (i), and having the same function as the amino acid sequence in (i); or (iii) an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or 100% sequence identity with the amino acid sequence as described in (i) or (ii). The polypeptide disclosed in this application has a significant inhibitory effect on cancer cells, low toxicity and a high safety index, and has the potential to prepare drugs for treating and / or preventing cancer.

Description

Technical Field

[0001] The present application relates to the field of biomedical technologies, and particularly to a polypeptide for inhibiting the expression of glutamine transporter II, and a preparation method and application thereof. Background Art

[0002] The abnormal metabolic characteristics of tumor cells have attracted increasing attention as new therapeutic targets. In tumor cells, the abnormally increased glutamine not only provides necessary nutrients and precursors for macromolecule synthesis for the cells, but also plays an important role in maintaining cellular oxidative balance, mitochondrial membrane stability, and mediating the mTOR signaling pathway. Glutamine transporter II (ASCT2) is a Na + -dependent neutral amino acid transporter encoded by the SLC1A5 gene, which is mainly located in the cell membrane and participates in mediating the exchange of amino acid substrates, and is the first checkpoint for glutamine to enter the cell.

[0003] Research has found that the expression level of ASCT2 is significantly up-regulated in liver cancer cells, and its expression level is negatively correlated with cancer prognosis, indicating that liver cancer cells need to increase glutamine metabolism by increasing the expression level of ASCT2 to maintain the continuous supply of nutrients required for their rapid proliferation, and such a large-scale energy metabolism demand rarely occurs in normal cells. Currently, small molecule inhibitors developed for the ASCT2 target show significant inhibitory effects on tumor cells, but have the disadvantages of certain potential toxicity, weak affinity, and easy generation of drug resistance.

[0004] Therefore, the existing technology still needs to be improved. Summary of the Invention

[0005] In view of the deficiencies of the above-mentioned existing technology, the purpose of the present application is to provide a polypeptide for inhibiting the expression of glutamine transporter II, and a preparation method and application thereof, aiming to solve the problems of potential toxicity, weak affinity, and easy generation of drug resistance of small molecule ASCT2 inhibitors.

[0006] The technical solution of the present application is as follows:

[0007] In the first aspect of the present application, there is provided a polypeptide for inhibiting the expression of glutamine transporter II, and the polypeptide has:

[0008] (i) an amino acid sequence as shown in SEQ ID No.1; or

[0009] (ii) an amino acid sequence obtained by substituting, deleting, or adding one or more amino acids to the amino acid sequence as described in (i), and having the same function as the amino acid sequence as described in (i); or

[0010] (iii) An amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or 100% sequence identity with the amino acid sequence described in (i) or (ii).

[0011] Optionally, the average molecular weight of the polypeptide is 2045 g / mol, the extinction coefficient of the polypeptide is 5690 M -1 cm -1 , and the theoretical isoelectric point pH value of the polypeptide is 11.48.

[0012] In a second aspect of the present application, there is provided a method for preparing a polypeptide that inhibits the expression of glutamine transporter II provided in the present application, comprising the steps of:

[0013] Obtaining the amino acid sequence of the polypeptide that inhibits the expression of glutamine transporter II through rational molecular design;

[0014] Synthesizing the polypeptide that inhibits the expression of glutamine transporter II using solid-phase synthesis.

[0015] Optionally, the rational molecular design is based on cell-penetrating peptides.

[0016] In a third aspect of the present application, there is provided an application of the polypeptide that inhibits the expression of glutamine transporter II provided in the present application in the preparation of a drug for treating and / or preventing cancer.

[0017] Optionally, the cancer is liver cancer, breast cancer, rectal cancer or lung cancer.

[0018] In a fourth aspect of the present application, there is provided a pharmaceutical composition for treating and / or preventing cancer, comprising the polypeptide that inhibits the expression of glutamine transporter II provided in the present application.

[0019] Optionally, the composition comprises a pharmaceutically acceptable excipient.

[0020] Advantages of the present application:

[0021] (1) The polypeptide that inhibits the expression of glutamine transporter II provided in the present application has a significant effect of inhibiting the expression of glutamine transporter II, inhibits the metabolism of glutamine in cancer cells or tumor cells, and has an inhibitory effect on cancer cells or tumor cells.

[0022] (2) The polypeptide that inhibits the expression of glutamine transporter II provided in the present application is designed based on cell-penetrating peptides, avoiding the drug resistance barrier of small molecule inhibitors of glutamine transporter II in the prior art, not easily generating drug resistance, and at the same time, as an amino acid, having low toxicity and great potential in the preparation of drugs for treating and / or preventing cancer. Description of the Drawings

[0023] To more clearly illustrate the technical solutions in the embodiments of the present application, the accompanying drawings required for the description of the embodiments will be briefly introduced below.

[0024] Figure 1 Liquid chromatography analysis chart of the polypeptide for inhibiting the expression of glutamine transporter II provided by the embodiment of the present application;

[0025] Figure 2 Liquid chromatography analysis chart of the polypeptide for inhibiting the expression of glutamine transporter II provided by the embodiment of the present application;

[0026] Figure 3 Cell plate counting chart provided by the embodiment of the present application;

[0027] Figure 4 Cell viability analysis chart provided by the embodiment of the present application. Detailed implementation manners

[0028] To make the objectives, technical solutions, and advantages of the present application clearer, the technical solutions in the embodiments of the present application will be clearly and completely described below in conjunction with the accompanying drawings and embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments of the present application, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present application. Without conflict, the following embodiments and the features in the embodiments can be combined with each other.

[0029] It should be noted that if there are descriptions involving "first", "second", etc. in the embodiments of the present application, such "first", "second", etc. descriptions are only for descriptive purposes and should not be construed as indicating or implying their relative importance and implicitly indicating the quantity of the indicated technical features. Thus, the features defined with "first", "second" may explicitly or implicitly include at least one of such features. In addition, the technical solutions between various embodiments can be combined with each other, but it must be based on the fact that those of ordinary skill in the art can implement it. When the combination of technical solutions conflicts or cannot be implemented, it should be considered that such a combination of technical solutions does not exist and is not within the protection scope required by the present application.

[0030] In the prior art, by developing small molecule inhibitors targeting the glutamine transporter II (ASCT2) target, the expression of ASCT2 is inhibited to inhibit the development of cancer cells. However, small molecule inhibitors are prone to drug resistance, have potential toxicity, and weak affinity. Based on this, in the first aspect of the embodiments of the present application, a polypeptide for inhibiting the expression of glutamine transporter II is provided, having:

[0031] (i) an amino acid sequence as shown in SEQ ID No. 1; or

[0032] (ii) An amino acid sequence obtained by substituting, deleting, or adding one or more amino acids to the amino acid sequence of (i), and having the same function as the amino acid sequence of (i); or

[0033] (iii) An amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity with the amino acid sequence of (i) or (ii).

[0034] It should be noted that "sequence identity" refers to the degree of identity between any given query sequence and subject sequence. Those skilled in the art will readily understand how to determine the identity between two polypeptides (e.g., an unmodified peptide and a peptide variant). For example, identity can be calculated after aligning the two sequences to achieve the highest level of identity, such as introducing gaps. Another method for calculating identity can be implemented through publicly available algorithms.

[0035] In addition, when determining the degree of sequence identity between two amino acid sequences, those skilled in the art can consider so-called "conservative" amino acid substitutions, which can generally be described as amino acid substitutions in which an amino acid residue is replaced with another amino acid residue having a similar chemical structure, and which have little or no effect on the function, activity, or other biological properties of the polypeptide. Such conservative amino acid substitutions are well known in the art.

[0036] In one embodiment, the average molecular weight of the polypeptide is 2045 g / mol, the extinction coefficient of the polypeptide is 5690 M -1 cm -1 , and the theoretical isoelectric point pH value of the polypeptide is 11.48.

[0037] In a second aspect of the embodiments of the present application, a method for preparing a polypeptide that inhibits the expression of glutamine transporter II is provided, including the steps of:

[0038] Obtaining the amino acid sequence of the polypeptide that inhibits the expression of glutamine transporter II through rational molecular design;

[0039] Synthesizing the polypeptide that inhibits the expression of glutamine transporter II using solid-phase synthesis.

[0040] Among them, the specific steps for obtaining the amino acid sequence of the polypeptide that inhibits the expression of glutamine transporter II through rational molecular design include:

[0041] Rational molecular design is based on cell-penetrating peptides. Through the spatial structure analysis of ASCT2, a hydrophobic pocket composed of several amino acids is found in the middle region of the stem of the ASCT2 structure. Acting on this region can cause conformational changes in ASCT2, thereby inhibiting the transport process of glutamine. For this key binding site, based on the structure of cell-penetrating peptides, the amino acid sequence of the polypeptide that inhibits the expression of glutamine transporter II is rationally designed (as shown in SEQ ID No.1).

[0042] Specifically, the anti-tumor activity of the target polypeptide is a balanced system jointly determined by various physicochemical constants. The factors affecting its activity mainly include hydrophilicity balance, charge, hydrophobicity, molecular size, spatial arrangement of groups, etc. By comprehensively evaluating the above parameters and examining the variation ranges of these properties, the envisioned ideal polypeptide sequence should have the following characteristics: less than 20 amino acids in length, mostly basic, with more than 2 positive charges and more than 30% hydrophobic amino acids, showing amphipathic structural characteristics in an environment similar to the cell membrane or the cell membrane, that is, one side is positively charged and the other side is hydrophobic. In addition, the N-terminus is rich in hydrophilic basic amino acid residues such as lysine and arginine; the C-terminus is amidated and rich in hydrophobic amino acids. The positively charged basic part promotes the interaction between the polypeptide and the target cell membrane, making it easy to adsorb to the cell membrane surface; the hydrophobic end is conducive to the polypeptide entering the lipid bilayer of the membrane and forming a specific structure.

[0043] In one embodiment, the amino acid sequence of the polypeptide that inhibits the expression of glutamine transporter II is obtained by combining rational molecular design and amino acid scanning. By combining rational molecular design and amino acid scanning, the amino acid sequence of the polypeptide can be predicted more accurately.

[0044] Solid-phase synthesis is a conventional technical means for polypeptide synthesis. Compared with liquid-phase synthesis and biosynthesis, solid-phase synthesis has relatively simple steps, high yield, and is suitable for large-scale production.

[0045] In the third aspect of the embodiments of the present application, there is provided an application of the polypeptide that inhibits the expression of glutamine transporter II in the above embodiments in the preparation of a drug for treating and / or preventing cancer. The polypeptide that inhibits the expression of glutamine transporter II inhibits the metabolism of glutamine in cancer cells or tumor cells by inhibiting the expression of ASCT2, and has an inhibitory effect on cancer cells or tumor cells. Therefore, it has the potential application in the preparation of a drug for treating and / or preventing cancer.

[0046] It should be noted that "prevention" refers to the preventive treatment of subclinical disease states, aiming to reduce the probability of the occurrence of clinical disease states. Prevention can be divided into primary prevention and secondary prevention. Primary prevention is defined as the treatment of subjects who have not yet presented with clinical disease states, while secondary prevention is defined as the prevention of the recurrence of the same or similar clinical disease states. "Treatment" refers to the treatment of diseases, symptoms or disorders, including: inhibiting the development of diseases, symptoms or disorders and / or delaying or alleviating diseases, symptoms or disorders.

[0047] In one embodiment, the cancer includes liver cancer, breast cancer, colon cancer, rectal cancer or lung cancer. It can be understood that the drugs for treating and / or preventing cancer may have the same or similar effects on liver cancer cells, breast cancer cells, rectal cancer cells, colon cancer cells, lung cancer cells as they have on liver cancer, breast cancer, colon cancer, rectal cancer or lung cancer, and also have the same or similar effects on tumor cells in the liver, on the breast, on the rectum, on the colon, and in the lungs.

[0048] In the fourth aspect of the embodiments of the present application, there is provided a pharmaceutical composition for treating and / or preventing cancer, comprising the polypeptide that inhibits the expression of glutamine transporter II in the above embodiments.

[0049] In one embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient. Pharmaceutically acceptable excipients include but are not limited to at least one of pharmaceutical carriers, diluents, adjuvants, and excipients.

[0050] Further illustration will be made through specific examples below.

[0051] Unless otherwise specified, the raw materials, reagents or devices used in the following examples can be obtained from conventional commercial channels or can be obtained by existing known methods.

[0052] The required experimental materials are:

[0053] Liver cancer cells (SNU449 / HepG2 / SK-Hep1) and normal liver tissue cell lines (L02) were purchased from the Cell Resource Center of the Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences; liver cancer cells (JHH1 / HUH7) and mouse peritoneal mononuclear macrophages (RAW264.7) were purchased from Wuhan Punosai Life Science and Technology Co., Ltd.

[0054] The required experimental reagents are:

[0055] The full set of Fmoc-protected amino acids, solid-phase resin and condensing agents (HBTU, HOBt) were purchased from Gil Biochemical Co., Ltd. (Shanghai, China); piperidine was purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China); ninhydrin, trifluoroacetic acid, 1,2-ethanedithiol, thioanisole, chromatographically pure acetonitrile, and chromatographically pure methanol were purchased from Aladdin Biochemical Technology Co., Ltd. (Shanghai, China); DMEM high-glucose medium, 1640 medium, fetal bovine serum, and trypsin were purchased from Gibco (USA); the CCK-8 kit was purchased from Dojindo Laboratories (Japan); the MTT reagent was purchased from Shanghai Macklin Biochemical Co., Ltd.; V-9302 (ASCT2 inhibitor) was purchased from Shanghai Yuanye Bio-Technology Co., Ltd.

[0056] Example 1: Synthesis of Polypeptide

[0057] The polypeptide was synthesized using solid-phase synthesis of amide MHBA resin and standard 9-fluorenylmethoxycarbonyl (Fmoc) amino acids. The polypeptide elongation reaction conditions were as follows: standard HBTU / HOBt as the condensing agent, N,N-dimethylformamide as the solvent, 10-fold excess of diisopropylethylamine, 3-fold excess of Fmoc-protected group amino acids or 5-fold excess of free fatty acids. The polypeptide was cleaved from the resin using a reagent composed of 87.5% trifluoroacetic acid, 2.5% ethanedithiol, 5% thioanisole, and 5% deionized water, and reacted at room temperature for 3 hours. Then the polypeptide was precipitated in methyl tert-butyl ether - petroleum ether (volume ratio 1:1) and air-dried overnight. The next day, the air-dried polypeptide was successively placed in a rotary evaporator and a vacuum drying oven to be dried, and stored refrigerated. The polypeptide was named FLK-18.

[0058] The molecular weight of FLK-18 was confirmed by electrospray ionization mass spectrometry, and the measured molecular weight of FLK-18 was 2045 g / mol. The purity of FLK-18 was analyzed by HPLC on a C18 column (250×4.6 mm), and the mobile phase was composed of solvent A (water containing 0.075% trifluoroacetic acid) and solvent B (methanol containing 0.075% trifluoroacetic acid). Gradient: 15% to 20% B for 2 minutes, 20% to 60% B for 6 minutes, 60% to 80% B for 4 minutes, 80% to 90% B for 4 minutes. The HPLC analysis results are as Figure 1 and Figure 2 shown, ensuring that the sample purity is greater than 95%, meeting the basic conditions for subsequent research.

[0059] Example 2: Determination of the Half-maximal Inhibitory Concentration of the Polypeptide against Hepatocarcinoma Cells

[0060] The CCK-8 method was used for determination. Different hepatocarcinoma cells (Huh-7, HepG2, SMMC-7721 / V9302, approximately 5×10 4Cells (1×10⁴ cells / well) were seeded in 96-well plates. After growing overnight, fresh medium containing different gradient concentrations of the test samples was added and incubated for 48 hours. The test samples included FLK-18 synthesized in Example 1 and four other similar polypeptides, namely FLS-18, FFG-17, FLK-14, and LFK-10. Then, 10 μL of CCK-8 was added to each well and incubated at 37 °C for another 2 hours. The absorbance was measured at 450 nm using a microplate reader, and the cytotoxicity was evaluated by comparing the growth inhibition rate of cells after treatment with the samples. The inhibition rate of untreated control cells was set at 0%. The half-maximal inhibitory concentration (IC₅₀) is shown in Table 1.

[0061] Table 1. Half-maximal inhibitory concentration of several polypeptides against liver cancer cells

[0062] Polypeptide number Huh-7 HepG2 SMMC7721 / V9302 FLS-18 24.51±3.44 40.27±2.58 35.19±1.92 FLK-18 5.61±0.22 0.75±0.71 3.85±0.03 FFG-17 7.26±0.31 4.49±0.59 10.85±1.13 FLK-14 127.33±6.51 83.52±4.39 201.27±3.2 LFK-10 327±10.24 104±3.35 151±6.72

[0063] As can be seen from Table 1, FLK-18 showed significant inhibitory effects on the proliferation of liver cancer cells in vitro (IC₅₀ = 0.75 - 5.61 μM), and it still exerted a therapeutic effect against clinically drug-resistant strains that were ineffective against the existing small molecule ASCT2 inhibitor V-9302. This indicates that FLK-18 has obvious inhibitory effects on the proliferation of liver cancer cells and also has inhibitory effects on liver cancer cells that are resistant to small molecule ASCT2 inhibitors. 50

[0064] Example 3: Comparison of the inhibitory effects of polypeptides on liver cancer cells

[0065] The plate colony formation assay was used. Glutamine-dependent cells (SNU449 / HepG2 / SK-Hep1) and glutamine-independent liver cancer cells (JHH1 / HUH7) were separately selected and seeded into 6-well plates at a density of approximately 200 cells per well. The next day, the cells were treated with different gradient concentrations of the test samples for 24 hours, and then the growth medium was replaced. After 8 days, the cell colonies were fixed with trypan blue staining solution (75% methanol / 25% acetic acid / 0.25% trypan blue), washed, air-dried, and the colonies were counted. The results are as Figure 3 shown.

[0066] From Figure 3 this, it can be seen that FLK-18 has significant inhibitory effects on both glutamine-dependent liver cancer cells (SNU449 / HepG2 / SK-Hep1) and glutamine-independent liver cancer cells (JHH1 / HUH7), while the ASCT2 inhibitor V-9302 is only effective against glutamine-dependent cells. It is further speculated that FLK-18 may have a synergistic inhibitory effect on the proliferation of liver cancer cells through multiple targets.

[0067] Example 4: Determination of the activity of polypeptides against normal cells

[0068] The safety of FLK-18 was verified through cell viability experiments. Human normal liver cells L02 and mouse peritoneal macrophages RAW264.7 were inoculated in 96-well plates at a density of approximately 8,000 cells per well. After the cells adhered overnight, they were replaced with 1640 medium containing different concentrations of FLK-18 and incubated for another 24 hours. 10 μL of MTT solution (5 mg / mL) was added to each well and the cells were cultured for an additional 4 hours. Then, the supernatant was carefully aspirated after centrifugation, and 100 μL of dimethyl sulfoxide was added to each well. The plate was placed on a shaker and shaken at low speed for 10 min to fully dissolve the crystals. The absorbance of each well was measured at 490 nm. The results are as Figure 4 shown.

[0069] As Figure 4 can be seen, after FLK-18 acted on the two normal cell lines (L02 and RAW 264.7) for 48 hours, the IC 50 values were both above 160 μM, far higher than its pharmacodynamic dose, indicating a high safety index. It can be considered that its inhibitory effect on cells is selective and it is basically non-toxic to normal cells.

[0070] In summary, the polypeptide provided in this application for inhibiting the expression of glutamine transporter II has a significant effect on inhibiting the expression of glutamine transporter II, inhibits the metabolism of glutamine in cancer cells or tumor cells, and has an inhibitory effect on cancer cells or tumor cells. At the same time, the polypeptide provided in this application for inhibiting the expression of glutamine transporter II is designed based on cell-penetrating peptides, circumventing the drug resistance barrier of small molecule inhibitors of glutamine transporter II in the prior art and being less likely to develop drug resistance. Moreover, as an amino acid, it has low toxicity and great potential in the preparation of drugs for treating and / or preventing cancer. In addition, the polypeptide provided in this application for inhibiting the expression of glutamine transporter II is synthesized by solid-phase synthesis, which is convenient, fast, stable, controllable, and has low production costs.

[0071] It should be understood that the applications of this application are not limited to the above examples. For those of ordinary skill in the art, improvements or changes can be made based on the above description, and all such improvements and changes should fall within the protection scope of the appended claims of this application.

Claims

1. A polypeptide that inhibits the expression of glutamine transporter II, characterized in that, The polypeptide has an amino acid sequence as shown in SEQ ID No.

1.

2. The polypeptide according to claim 1, characterized in that, The molecular weight of the polypeptide is 2045 g / mol.

3. Use of a polypeptide for inhibiting the expression of glutamine transporter II as claimed in claim 1 in the preparation of a medicament for inhibiting the proliferation of liver cancer cells.

4. A pharmaceutical composition for inhibiting the proliferation of liver cancer cells, characterized in that, Comprising the polypeptide for inhibiting the expression of glutamine transporter II as claimed in claim 1.

5. The composition according to claim 4, characterized in that The composition comprises a pharmaceutically acceptable excipient.

Images