A polypeptide with inhibitory activity against triple-negative breast cancer cells and its application

Abstract

The present invention belongs to the field of biomedicine, and particularly relates to a polypeptide with inhibitory activity against triple-negative breast cancer cells and its application. The amino acid sequence of the polypeptide is shown as SEQ ID NO.1 to SEQ ID NO.14. The polypeptide of the present invention can be obtained by solid-phase synthesis, has a high inhibitory activity against human breast cancer cell MDA-MB-231, and a low inhibition rate against normal cell MCF 10A cells.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, specifically relates to the field of polypeptide technology, and more specifically relates to a polypeptide with inhibitory activity against triple-negative breast cancer cells and its application. Background Art

[0002] Breast cancer is the most common type of female malignant tumor in the world. Clinically, the main indicators for determining the pathological type of breast cancer are estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor-2 (HER2). Classification is based on the positive or negative of these three indicators. When the pathological report shows that all three of the above indicators are negative, it is clinically determined as triple-negative breast cancer (TNBC). In the past decade, the proportion of triple-negative breast cancer has been gradually increasing and currently accounts for about 15% - 20% of all breast cancer pathological types.

[0003] Triple-negative breast cancer has the following characteristics: it occurs frequently in young women, usually women under 40 years old; it has a high recurrence rate, and there is a recurrence peak within 1 - 3 years after surgery; it has a high invasiveness, with a large tumor size and a high degree of malignancy, and is commonly found in the lungs, brain, and liver; it is insensitive to conventional treatments and is prone to drug resistance, resulting in a poor prognosis for patients, with a median survival period of only 13 months. Triple-negative breast cancer has now become the focus of breast cancer research and attention in recent years.

[0004] Under normal circumstances, when the estrogen receptor and progesterone receptor are expressed positively, hormone endocrine regulation therapy can be used; for breast cancer with positive HER2, anti-HER2 targeted therapy can be used. However, all three indicators of triple-negative breast cancer are negative, lacking endocrine and anti-HER2 treatment targets, so neither endocrine therapy nor targeted therapy can be used, which is therefore a treatment difficulty.

[0005] The overall survival of untreated triple-negative breast cancer is 9 months, and that of advanced patients is about half a year. Even after treatment, the overall survival is only a little over one year. Currently, the treatment methods for breast cancer include surgical treatment, neoadjuvant chemotherapy, adjuvant chemotherapy, endocrine therapy, and targeted therapy, etc. Since triple-negative breast cancer lacks specific molecular markers, the current first-line adjuvant treatment plan in clinical practice is still mainly chemotherapy, lacking specific treatment means. Clinically, intervention is often carried out by combining drugs. The combined chemotherapy of taxanes and anthracyclines is the standard treatment method for early triple-negative breast cancer patients, but it increases the risk of cardiac mortality, secondary leukemia, and myelodysplastic syndrome to a certain extent. Carboplatin increases the incidence of neutropenia and thrombocytopenia, while bevacizumab can cause hypertension, infection, thromboembolism, bleeding, and postoperative complication events.

[0006] Patent CN110950931A describes a polypeptide specifically targeting triple-negative breast cancer stem cells and its application, and patent CN111018951A describes a polypeptide targeting triple-negative breast cancer cells and its application.

[0007] In the case where the effect of traditional treatment methods is limited, the role of innovative drugs is becoming more and more important. In recent years, immunotherapy drugs and neoadjuvant therapies and their drugs are also under research and development. It is expected that the emergence of innovative drugs can bring new hope to triple-negative breast cancer patients. In summary, it is of great significance to study effective drugs for triple-negative breast cancer. Summary of the Invention

[0008] To solve the deficiencies of the prior art, the present invention provides a polypeptide having an inhibitory activity on triple-negative breast cancer cells and its application.

[0009] On the one hand, the present invention provides a polypeptide, the amino acid sequence of which is as shown in SEQ ID NO.1 to SEQ ID NO.2 or SEQ ID NO.3 to SEQ ID NO.14, wherein the first and last amino acids of SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.13, and SEQ ID NO.14 form a ring through a peptide bond; the amino acid sequence SEQ ID NO.3 to SEQ ID NO.14 is a linear peptide or cyclic peptide obtained by disassembling the amino acid sequence SEQ ID NO.1 or SEQ ID NO.2.

[0010] Among them, the 80-amino acid cyclic peptide with the amino acid sequence as shown in SEQ ID NO.1 or SEQ ID NO.2 is an 80-cyclic peptide detected by high-throughput screening technology from a polypeptide library and having an inhibitory activity on triple-negative breast cancer cells. The discovery of this cyclic peptide includes dissolving and diluting the polypeptide library, and detecting OD in an enzyme-linked immunosorbent assay 450 , according to OD450 The method for calculating the inhibition rate is screened from a polypeptide library.

[0011] Furthermore, the polypeptide provided by the present invention has an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 1 to SEQ ID NO: 14 or a derivative of the amino acid sequence shown by SEQ ID NO: 1 to SEQ ID NO: 14 that has been modified.

[0012] In some embodiments, the derivative of the modified amino acid sequence is selected from one or more of the following modifications: N-terminal and / or C-terminal modification; substitution of one or more amino acid residues with one or more natural and / or non-natural amino acid residues.

[0013] In some embodiments, the modifications include amination, hydroxylation, carboxylation, carbonylation, amidation, alkylation, phosphorylation, glycosylation, cyclization, biotinylation, acetylation, esterification, fluorescent group modification, polyethylene glycol (PEG) modification, immobilization modification.

[0014] In some embodiments, the amino acid sequences SEQ ID NO. 3 to SEQ ID NO. 14 are linear peptides or cyclic peptides obtained by disassembling SEQ ID NO. 1 or SEQ ID NO. 2, wherein the length of the linear peptide is 22 to 32 amino acids or 35 to 45 amino acids; the length of the cyclic peptide is 30 to 40 amino acids.

[0015] In some embodiments, the length of the linear peptide is 22 to 32 amino acids or 35 to 45 amino acids.

[0016] In some embodiments, the length of the linear peptide is 31 amino acids.

[0017] In some embodiments, the length of the linear peptide is 40 amino acids.

[0018] In some embodiments, the polypeptide is a cyclic peptide obtained by analyzing and disassembling SEQ ID NO. 1 or SEQ ID NO. 2, and the first amino acid and the last amino acid form a ring through a peptide bond.

[0019] In some embodiments, the length of the cyclic peptide is 30 to 40 amino acids.

[0020] In some embodiments, the length of the cyclic peptide is 33 amino acids.

[0021] On the other hand, the present invention provides a polynucleotide molecule, which comprises one or two polynucleotide molecules capable of encoding the above-mentioned polypeptide.

[0022] Furthermore, the polynucleotide molecule comprises a polynucleotide molecule capable of encoding the polypeptides shown in SEQ ID NO.1 to SEQ ID NO.14.

[0023] On the other hand, the present invention provides a pharmaceutical composition, which comprises a therapeutically effective amount of the above polypeptide or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

[0024] On the other hand, the present invention provides the use of the polypeptide or a pharmaceutically acceptable salt thereof, the polynucleotide molecule and the pharmaceutical composition in the preparation of a drug for treating breast cancer.

[0025] Furthermore, the application is for triple-negative breast cancer.

[0026] Term

[0027] Unless otherwise defined herein, scientific and technical terms used in this patent application shall have the meanings commonly understood by those of ordinary skill in the art.

[0028] As used herein, the "peptide library" is a peptide library constructed by Hunan Zhongcheng Quanta Biochemical Co., Ltd. using the PICT (Peptide Information Compression Technology) patent technology. This technology compresses peptide information by biological means, integrating the information of multiple peptides into one peptide, thereby achieving a relatively small library capacity containing a large amount of peptide information; a cyclic peptide library containing nearly 73,000 80-amino acid peptides is constructed by the PICT technology. The specific construction method can be found in Patent CN201580081102.3 and Patent CN201780089941.9.

[0029] The pharmaceutical composition used in the method of the present invention may contain any pharmaceutically acceptable excipient. Examples of excipients include, but are not limited to, starch, sugar, microcrystalline cellulose, diluent, granulating agent, lubricant, binder, disintegrant, wetting agent, emulsifier, coloring agent, release agent, coating agent, antioxidant, plasticizer, gelling agent, thickening agent, hardening agent, coagulant, suspension agent, surfactant, humectant, carrier, stabilizer, and combinations thereof.

[0030] The pharmaceutical composition used in the method of the present invention may contain any pharmaceutically acceptable carrier. For example, the carrier may be a liquid or solid filler, diluent, excipient, solvent, or encapsulating substance, or combinations thereof.

[0031] In various embodiments, the pharmaceutical composition of the present invention can be formulated for delivery by any route of administration. This may include, for example, aerosol, nasal, oral, transmucosal, transdermal, parenteral, or enteral.

[0032] "Parenteral" refers to administration routes typically associated with injection, including intraorbital, infusion, intraarterial, intracapsular, intracardiac, intradermal, intramuscular, intraperitoneal, intralung, intraspinal, intrasternal, intrathecal, intrauterine, intravenous, subarachnoid, subcapsular, subcutaneous, transmucosal or transtracheal. By the parenteral route, the composition can be in the form of a solution or suspension for infusion or for injection, or in the form of a lyophilized powder. By the parenteral route, the composition can be in the form of a solution or suspension for infusion or for injection. By the enteral route, the pharmaceutical composition can be in the form of tablets, gel capsules, sugar-coated tablets, syrups, suspensions, solutions, powders, granules, emulsions, microspheres or nanospheres or lipid vesicles or polymer vesicles allowing controlled release. Generally, the composition is administered by injection. The methods for such administrations are known to those skilled in the art.

[0033] Regarding sequence identity. Sequence identity is calculated by sequence alignment according to methods known in the art. To determine the percentage identity of two amino acid sequences, the sequences are aligned for optimal comparison. For example, gaps can be introduced in the sequence of the first amino acid sequence for optimal alignment with the second amino acid sequence. Then the amino acid residues at the corresponding amino acid positions are compared. When the position in the first sequence is occupied by the same amino acid residue as at the corresponding position in the second sequence, the molecules are identical at that position. The percentage identity between two sequences is a function of the number of identical positions shared by the sequences. Thus % identity = number of identical positions / total number of overlapping positions multiplied by 100. In this comparison, the sequences can be of the same length or can be of different lengths. The optimal sequence alignment for determining the comparison window can be carried out by the local homology algorithm of Smith and Waterman (J. Theor. Biol., 1981), by the homology alignment algorithm of Needleman and Wunsch (J. Mol. Biol, 1972), by the method of Pearson and Lipman for finding similarity (Proc. Natl. Acad. Sci. U.S.A., 1988), by the computerized implementation of these algorithms (GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics Software Package version 7.0, Genetic Computer Group, 575, Science Drive, Madison, Wisconsin) or for example using publicly available computer software such as BLAST. When using such software, it is preferred to use the default parameters, such as the gap penalty or the extension penalty. The best alignment produced by the various methods is selected (i.e., the alignment that produces the highest percentage identity over the entire comparison window).

[0034] Compared with the prior art, the present invention has the following advantages:

[0035] (1) The polypeptide library used in the present invention is more efficient compared to traditional polypeptide screening. Each compound in the library is independently produced, and all have been identified by mass spectrometry and accurately weighed, ensuring the accuracy and stability of screening and avoiding the distortion problem (the actual library capacity is much lower than the theoretical value) of traditional phage libraries and other mixed compound libraries. Compounds in the library can be screened either mixed or individually, with diverse and flexible screening methods, avoiding the mutual interference of each component during the screening of pure mixture libraries.

[0036] (2) The polypeptide inhibitor provided by the present invention is inexpensive and can be synthesized on a large scale by chemical methods; it has high stability and can be stored at room temperature for a long time in freeze-dried form; it has extremely low immunogenicity.

[0037] (3) The present invention provides a polypeptide with inhibitory activity against triple-negative breast cancer cells, which has high inhibitory activity against human breast cancer cell MDA-MB-231 and low inhibitory rate against normal cell MCF 10A cells. Description of the Drawings

[0038] Figure 1 It is the concentration-response result of SEQ ID NO.1 to SEQ ID NO.2 in Example 1;

[0039] Figure 2 It is the concentration-response result of SEQ ID NO.3 to SEQ ID NO.6 in Example 2;

[0040] Figure 3 It is the concentration-response result of SEQ ID NO.7 to SEQ ID NO.10 in Example 2;

[0041] Figure 4 It is the concentration-response result of SEQ ID NO.11 to SEQ ID NO.14 in Example 2. Detailed Embodiments

[0042] To better understand the present invention, the following further describes the present invention in detail with reference to the drawings and examples, but does not limit the present invention in any way. Any transformation or improvement based on the teachings of the present invention falls within the protection scope of the present invention.

[0043] The key reagents required for the present invention are: polypeptide library (self-made), triple-negative breast cancer cell line MDA-MB-231 (Procell CL-0150A), human normal mammary epithelial cell MCF 10A (Procell CL-0525).

[0044] The polypeptide compounds and their derivatives provided by the present disclosure are synthesized by solid-phase synthesis. The synthesis carrier is Fmoc-Cys(Trt)-2-Chlotrityl Resin. During the synthesis process, first, the Fmoc-Cys(Trt)-2-Chlotrityl Resin is fully swollen in N,N-dimethylformamide (DMF), and then the solid-phase carrier and the activated amino acid derivatives are repeatedly subjected to the operations of condensation → washing → deprotection of Fmoc → washing → the next round of amino acid condensation to reach the desired polypeptide chain length. Finally, a mixed solution of trifluoroacetic acid: water: triisopropylsilane: benzyl methyl sulfide (90:2.5:2.5:5, v:v:v:v) is reacted with the resin to cleave the polypeptide from the solid-phase carrier, and then the solid crude product of the linear precursor is obtained after precipitation with cold methyl tert-butyl ether. The crude linear precursor after cleavage is oxidized by disulfide bonds in an alkaline solution to obtain the crude target polypeptide. The crude polypeptide is purified and separated by a C-18 reversed-phase preparative chromatography column in a system of acetonitrile / water with 0.1% trifluoroacetic acid to obtain the pure product of the polypeptide and its derivatives. The obtained amino acid sequences are shown in Table 1.

[0045] Table 1 Amino acid sequences of SEQ ID NO.1 to SEQ ID NO.14

[0046]

[0047]

[0048] Example 1

[0049] 1. High-throughput screening process

[0050] 1.1. Culturing MDA-MB-231 and MCF 10A cells

[0051] 1.1.1. Cell resuscitation: Take out the cells from the liquid nitrogen tank and quickly thaw the cells in a 37°C water bath. Transfer the cells to a 15 mL centrifuge tube, slowly add 9 mL of preheated thawing medium, centrifuge at 800 rpm for 5 minutes, and remove the supernatant medium. Resuspend the cells with 5 mL of thawing medium, transfer them to a T25 culture flask, and culture them in an incubator at 37°C and 5% CO2. Replace the culture medium with growth medium on the second day after cell resuscitation.

[0052] 1.1.2. Cell passage: When the cells reach 80-90% confluence in the culture flask, first rinse the cells with DPBS, then add DPBS again and gently tap the cell flask to make the cells detach from the flask wall; collect the cell suspension into a centrifuge tube, centrifuge at 800 rpm for 5 minutes, and remove the supernatant medium; add 6-8 mL of fresh growth medium, resuspend the cells, and passage them at a ratio of 1:3 to 1:10, and culture them in an incubator at 37°C and 5% CO2. Change the medium every 2-3 days after passage.

[0053] 1.1.3. Cell cryopreservation: When the cells reach 80 - 90% confluence in the culture dish, first rinse the cells with DPBS, then re - add DPBS and gently tap the cell bottle to detach the cells from the wall; collect the cell suspension into a centrifuge tube, centrifuge at 800 rpm for 5 minutes, and remove the supernatant culture medium; resuspend the cells with cryopreservation medium, perform cell counting, and dilute the cells to 2 - 3×10 6 / mL. Aliquot 1 mL of the cell cryopreservation suspension into each cryotube. Place the cryotubes filled with cells into a cryobox, put the cryobox into an - 80 °C refrigerator for overnight storage, and then transfer the cryotubes to a liquid nitrogen tank.

[0054] 1.2 Seeding of MDA - MB - 231 and MCF 10A cells

[0055] The MDA - MB - 231 or MCF 10A cells in the culture flask are digested with 0.25% trypsin and suspended in cell culture medium. They are added to a 96 - well plate at a density of 10,000 cells per well using a pipettor, 100 μL per well, and cultured overnight at 37 °C in 5% CO₂.

[0056] 1.3 Screening of peptide library

[0057] After the cells are cultured overnight, add 10 μL of 100 μM peptide compound, and continue to culture in a 37 °C, 5% CO₂ incubator for 72 hours. On the day of detection, take out the cells, add 10 μL of CCK - 8 detection reagent, incubate at 37 °C for 3 hours, and detect OD 450 , calculate the inhibition rate, select the peptides with high inhibitory activity, and perform concentration - dependent testing.

[0058] 2. Experimental results

[0059] Through high - throughput screening, 2 peptides, SEQ ID NO.1 and SEQ ID NO.2, which can inhibit MDA - MB - 231 cells but have no inhibitory activity against MCF 10A cells, were found from nearly 73,000 80 - loop peptides. The inhibitory activities of SEQ ID NO.1 and SEQ ID NO.2 against MDA - MB - 231 cells were tested, and the experimental results are shown in Table 2 and Figure 1 as follows.

[0060] Table 2 Concentration - response results of SEQ ID NO.1 and SEQ ID NO.1

[0061] SEQ ID NO. <![CDATA[IC50 inhibition of MDA-MB-231 cells 50 (μM)]]> Inhibitory rate of 10 μM on MCF10A cells 1 0.51 -13.1％ 2 0.74 7.0％

[0062] Example 2

[0063] Screening of derivative peptides

[0064] 1. Screening and confirmation of polypeptides derived from SEQ ID NO.1 and SEQ ID NO.2

[0065] Using internal decompression technology, the amino acids of the 80-ring peptides (SEQ ID NO.1 and SEQ ID NO.2) were decompressed to design cyclic peptides or linear peptides with different amino acid sequences from 5 to 80. The decompressed polypeptides were screened according to the screening process in Example 1.

[0066] 2. Experimental results

[0067] The 80-ring peptides of SEQ ID NO.1 and SEQ ID NO.2 were decompressed to obtain a series of polypeptides with inhibitory activity against MDA-MB-231 cells. Their inhibitory activities against MDA-MB-231 cells were tested, and the experimental results are shown in Table 3 and Figures 2 to 4 as follows.

[0068] Table 3 Concentration-response results of SEQ ID NO.3 to SEQ ID NO.14

[0069]

[0070]

[0071] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application and not to limit the protection scope of the present application. Although the present application has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present application can be modified or equivalently replaced without departing from the essence and scope of the technical solutions of the present application.

Claims

1. A polypeptide, characterized in that, The amino acid sequence of the polypeptide is as shown in SEQ ID NO.2 or SEQ ID NO.14, wherein the first and last amino acids of the polypeptides shown in SEQ ID NO.2 and SEQ ID NO.14 form a ring through a peptide bond.

2. A polynucleotide molecule, characterized in that, The polynucleotide molecule comprises one or two polynucleotide molecules capable of encoding the polypeptide of claim 1.

3. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises a therapeutically effective amount of the polypeptide of claim 1 and a pharmaceutically acceptable carrier.

4. Use of the polypeptide according to claim 1, the polynucleotide molecule according to claim 2, or the pharmaceutical composition according to claim 3 in the preparation of a medicament for treating breast cancer, wherein the breast cancer is triple-negative breast cancer.

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