CN117285604A

CN117285604A - Polypeptide with inhibitory activity on triple negative breast cancer cells and application thereof

Info

Publication number: CN117285604A
Application number: CN202310903266.6A
Authority: CN
Inventors: 王登; 聂立波; 周贵仁; 刘惠清; 王芹芹; 王新波; 李振万; 董须丰; 李向群
Original assignee: Hohai University HHU
Current assignee: Hohai University HHU
Priority date: 2023-07-22
Filing date: 2023-07-22
Publication date: 2023-12-26

Abstract

The invention belongs to the field of biological medicine, and in particular relates to a polypeptide with inhibitory activity on triple negative breast cancer cells and application thereof. The polypeptide has an amino acid sequence shown in SEQ ID NO. 1-SEQ ID NO.14, can be obtained through solid phase synthesis, has high inhibition activity on human breast cancer cells MDA-MB-231, and has low inhibition rate on normal cells MCF 10A.

Description

Polypeptide with inhibitory activity on triple negative breast cancer cells and application thereof

Technical Field

The invention belongs to the field of biological medicine, in particular to the technical field of polypeptides, and more particularly relates to a polypeptide with inhibitory activity on triple negative breast cancer cells and application thereof.

Background

Breast cancer is one of the most common malignant tumors in women worldwide, and clinically, three main indicators of the pathological type of breast cancer are identified, namely an estrogen receptor (estrogen receptor, ER), a progestogen receptor (progesterone receptor, PR) and a human epidermal growth factor receptor 2 (human epithelial growth factor receptor-2, her 2), and are classified according to the positive or negative of the three indicators. When the pathology report shows that all three indexes are negative, the clinical diagnosis is three-negative breast cancer (triple negative breast cancer, TNBC). The proportion of triple negative breast cancers gradually rises in the last 10 years, and the proportion of triple negative breast cancers accounts for 15% -20% of all pathological types of breast cancers at present.

Triple negative breast cancer has the following characteristics: are frequent in young women, typically women under 40 years of age; the recurrence rate is high, and a recurrence peak appears between 1 and 3 years after the operation; high invasiveness, larger tumor, high malignancy, common lung, brain and liver; insensitive to conventional treatments and prone to developing drug resistance, resulting in a poor prognosis for patients with median survival of only 13 months. Triple negative breast cancer has now become the focus of research and attention for breast cancer in recent years.

In general, estrogen receptor and progestogen receptor expression are positive, and a mode of hormone endocrine modulation therapy can be adopted; HER2 positive breast cancer may be treated with anti-HER 2 targeting. However, three indexes of the triple-negative breast cancer are negative, and lack of endocrine and HER2 resisting treatment targets, so that endocrine treatment cannot be adopted, and targeted treatment cannot be adopted, so that the triple-negative breast cancer is a treatment difficulty.

The overall survival of untreated triple negative breast cancer is 9 months, and the total survival of advanced patients is about half a year, even though treated, is only one year. At present, the treatment means for the breast cancer comprise operation treatment, new auxiliary chemotherapy, endocrine treatment, targeted treatment and the like, and the clinical first-line auxiliary treatment scheme of the triple negative breast cancer still takes chemotherapy as the main treatment means and lacks specific treatment means due to the lack of specific molecular markers. Clinically, intervention is often performed by drug combination, and combination chemotherapy of taxane and anthracycline is the standard treatment for early triple negative breast cancer patients, but it increases to some extent the risk of heart mortality, secondary leukemias and myelodysplastic syndromes, carboplatin increases the incidence of neutropenia and thrombocytopenia, while bevacizumab causes hypertension, infections, thrombotic plugs, bleeding and postoperative complications.

Patent CN110950931a describes a polypeptide specifically targeting triple negative breast cancer stem cells and uses thereof, and patent CN111018951a describes a polypeptide targeting triple negative breast cancer cells and uses thereof.

Under the condition that the traditional treatment method has limited effect, the effect of innovative medicaments is more important. In recent years, immunotherapeutic drugs and neoadjuvant therapies, and drugs thereof, are also under development, and the advent of innovative drugs is expected to bring new light to triple negative breast cancer patients. In conclusion, the research on effective medicaments for triple negative breast cancer has important significance.

Disclosure of Invention

In order to solve the defects in the prior art, a polypeptide with an inhibitory activity on triple negative breast cancer cells and application thereof are provided.

In one aspect, the invention provides a polypeptide, the amino acid sequence of which is shown as SEQ ID NO. 1-SEQ ID NO.2 or SEQ ID NO. 3-SEQ ID NO.14, wherein the head and tail amino acids of SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.13 and SEQ ID NO.14 form a ring through peptide bonds; the amino acid sequences SEQ ID NO. 3-SEQ ID NO.14 are linear peptide or cyclic peptide obtained by disassembling the amino acid sequences SEQ ID NO.1 or SEQ ID NO.2.

Wherein the cyclic peptide with 80 amino acids and amino acid sequence shown as SEQ ID NO.1 or SEQ ID NO.2 is 80 cyclic peptide which is detected from polypeptide library by high throughput screening technology and has the effect of inhibiting the activity of triple negative breast cancer cells, the discovery of the cyclic peptide comprises dissolving and diluting the polypeptide library, and detecting OD in an enzyme-labeled instrument ₄₅₀ According to OD ₄₅₀ Value calculation inhibition ratioIs selected from a library of polypeptides.

Furthermore, the polypeptide provided by the invention has at least 95% of sequence identity with the amino acid sequence shown in SEQ ID NO. 1-SEQ ID NO.14 or modified derivatives of the amino acid sequence shown in the SEQ ID NO. 1-SEQ ID NO. 14.

In some embodiments, the derivative of the modified amino acid sequence is selected from one or more modifications of: n-terminal and/or C-terminal modification; one or more amino acid residues are substituted with one or more natural and/or unnatural amino acid residues.

In some embodiments, the modification comprises an amination, hydroxylation, carboxylation, carbonylation, amidation, alkylation, phosphorylation, glycosylation, cyclization, biotinylation, acetylation, esterification, fluorophore modification, polyethylene glycol PEG modification, immobilization modification.

In some embodiments, the amino acid sequences SEQ ID NO. 3-SEQ ID NO.14 are linear peptides or cyclic peptides obtained by resolution of the amino acid sequences SEQ ID NO.1 or SEQ ID NO.2, wherein the linear peptides are 22-32 amino acids or 35-45 amino acids in length; the cyclic peptide has a length of 30 to 40 amino acids.

In some embodiments, the linear peptide is 22 to 32 amino acids or 35 to 45 amino acids in length.

In some embodiments, the linear peptide is 31 amino acids in length.

In some embodiments, the linear peptide is 40 amino acids in length.

In some embodiments, the polypeptide is a cyclic peptide that is the result of analysis and resolution of SEQ ID NO.1 or SEQ ID NO.2, with amino acid 1 and the last 1 being cyclic via a peptide bond.

In some embodiments, the cyclic peptide is 30 to 40 amino acids in length.

In some embodiments, the cyclic peptide is 33 amino acids in length.

In another aspect, the invention provides a polynucleotide molecule comprising one or two polynucleotide molecules capable of encoding a polypeptide as described above.

Further, the polynucleotide molecule comprises a polynucleotide molecule capable of encoding the polypeptides shown in SEQ ID NO. 1-SEQ ID NO. 14.

In another aspect, the invention provides a pharmaceutical composition comprising a therapeutically effective amount of a polypeptide as described above, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

In another aspect, the invention provides the use of the polypeptide or a pharmaceutically acceptable salt, polynucleotide molecule and pharmaceutical composition thereof for the manufacture of a medicament for the treatment of breast cancer.

Further, the use is triple negative breast cancer.

Terminology

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

The polypeptide library is obtained by adopting PICT (Peptide Information Compression Technology) patent technology by the SangZhi full peptide Biochemical Co., hunan, and the technology compresses polypeptide information by using biological means, so that the information of a plurality of polypeptides can be integrated into one polypeptide, and the aim of containing larger polypeptide information with relatively smaller storage capacity is fulfilled; a cyclic peptide library containing approximately 73000 80 amino acids was constructed by the PICT technique. Specific construction methods can be seen in patent CN201580081102.3 and patent CN201780089941.9.

The pharmaceutical composition used in the methods of the invention may contain any pharmaceutically acceptable excipient. Examples of excipients include, but are not limited to, starches, sugars, microcrystalline cellulose, diluents, granulating agents, lubricants, binders, disintegrating agents, wetting agents, emulsifying agents, coloring agents, releasing agents, coating agents, antioxidants, plasticizers, gelling agents, thickening agents, hardening agents, solidifying agents, suspending agents, surfactants, humectants, carriers, stabilizers, and combinations thereof.

The pharmaceutical composition used in the methods of the invention may contain any pharmaceutically acceptable carrier. For example, the carrier may be a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, or a combination thereof.

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

By "parenteral" is meant a route of administration commonly associated with injection, including intraorbital, infusion, intraarterial, intracapsular, intracardiac, intradermal, intramuscular, intraperitoneal, intrapulmonary, intraspinal, intrasternal, intrathecal, intrauterine, intravenous, subarachnoid, subcapsular, subcutaneous, transmucosal, or transtracheal. By parenteral route, the compositions may be in the form of solutions or suspensions for infusion or for injection, or in the form of lyophilized powders. By parenteral route, the compositions may be in the form of solutions or suspensions for infusion or for injection. By the enteral route, the pharmaceutical composition may 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. Typically, the composition is administered by injection. Methods for such administration are known to those skilled in the art.

Regarding sequence identity. Sequence identity is calculated by sequence alignment according to methods known in the art. To determine the percent identity of two amino acid sequences, the sequences were 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. The amino acid residues at the corresponding amino acid positions are then compared. When a position in the first sequence is occupied by the same amino acid residue as the corresponding position in the second sequence, the molecules are identical at that position. The percent 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 may be the same length or may be different lengths. The optimal sequence alignment for determining the comparison window can be carried out by local homology algorithms of Smith and Waterman (j. Ther. Biol., 1981), by homology alignment algorithms of Needleman and Wunsch (j. Mol. Biol, 1972), by methods of Pearson and Lipman, searching for similarity (proc. Natl. Acad. Sci. U.S. A., 1988), by computerized implementation of these algorithms (GAP, BESTFIT, FASTA and TFASTA, genetic Computer Group,575,Science Drive,Madison,Wisconsin in the Wisconsin genetics software package 7.0) or e.g. using publicly available computer software such as BLAST. When such software is used, default parameters, such as gap penalties or extension penalties, are preferably used. The best alignment resulting from the various methods was selected (i.e., yielding the highest percent identity across the comparison window).

Compared with the prior art, the invention has the following advantages:

(1) Compared with the traditional polypeptide screening, the polypeptide library used in the invention has high efficiency, and each compound in the library is independently produced, and is identified and accurately weighed through mass spectrum, so that the screening accuracy and stability are ensured, and the problem of distortion (the actual library capacity is far lower than the theoretical value) of the traditional mixed compound library such as a phage library is avoided. The compounds in the library can be mixed or screened singly, the screening mode is various and flexible, and the mutual interference of each component in the screening of the pure mixture library is avoided.

(2) The polypeptide inhibitor provided by the invention has low price and can be synthesized in a large scale by a chemical method; the stability is high, and the product can be stored in a freeze-dried form at room temperature for a long time; the immunogenicity is extremely low.

(3) The invention provides a polypeptide with inhibitory activity on triple negative breast cancer cells, which has higher inhibitory activity on human breast cancer cells MDA-MB-231 and low inhibition rate on normal cells MCF 10A.

Drawings

FIG. 1 shows the concentration response of SEQ ID NO.1 to SEQ ID NO.2 in example 1;

FIG. 2 shows the concentration response of SEQ ID NO.3 to SEQ ID NO.6 in example 2;

FIG. 3 shows the concentration response of SEQ ID NO.7 to SEQ ID NO.10 in example 2;

FIG. 4 shows the concentration response of SEQ ID NO.11 to SEQ ID NO.14 in example 2.

Detailed Description

For a better understanding of the present invention, reference will now be made in detail to the present invention, examples of which are illustrated in the accompanying drawings and described in the accompanying drawings, wherein the present invention is not to be construed as in any way limiting, but any changes or modifications which are based on the teachings of the present invention are intended to fall within the scope of the invention.

The key reagents required by the invention are as follows: polypeptide library (homemade), triple negative breast cancer cell line MDA-MB-231 (Punuocele CL-0150A), human normal mammary epithelial cell MCF 10A (Punuocele CL-0525).

The polypeptide compound and the derivative thereof provided by the present disclosure are synthesized by a solid phase synthesis method. The synthetic carrier is Fmoc-Cys (Trt) -2-chloro Resin. In the synthesis process, fmoc-Cys (Trt) -2-chloro Resin is fully swelled in N, N-Dimethylformamide (DMF), then the solid phase carrier and the activated amino acid derivative are repeatedly condensed, washed, deprotected, fmoc-washed and the next round of amino acid condensation are performed to reach the length of the polypeptide chain to be synthesized, and finally trifluoroacetic acid is used for: water: triisopropylsilane: the mixed solution of the phenylsulfide (90:2.5:2.5:5:5, v: v: v) reacts with resin to crack the polypeptide from the solid phase carrier, and then the solid crude product of the linear precursor is obtained after the freezing methyl tertiary butyl ether sedimentation. And (3) performing disulfide bond oxidation on the cut linear precursor crude product in alkaline solution to obtain a target polypeptide crude product. Purifying and separating the crude polypeptide in acetonitrile/water system of 0.1% trifluoroacetic acid by C-18 reversed phase preparative chromatographic column to obtain pure product of polypeptide and its derivative. The amino acid sequences obtained are shown in Table 1.

TABLE 1 amino acid sequences of SEQ ID NO.1 to SEQ ID NO.14

Example 1

1. High throughput screening process

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

1.1.1. Cell resuscitation: the cells were removed from the liquid nitrogen tank and rapidly thawed in a 37 ℃ water bath. Cells were transferred to a 15mL centrifuge tube, 9mL of pre-warmed thawing medium was slowly added, centrifuged at 800 rpm for 5 minutes, and the supernatant medium was removed. The cells were resuspended in 5mL of thawing medium, transferred to T25 flasks, and placed at 37℃in 5% CO ₂ Is cultured in an incubator of (a). The following day of cell resuscitation the medium was replaced with growth medium.

1.1.2. Cell passage: when the cells grow up to 80-90% of the culture bottle, the cells are firstly rinsed by DPBS, and then the DPBS is added again to tap the cell bottle practice report to fall off from the bottle wall; collecting cell suspension into a centrifuge tube, centrifuging at 800 rpm for 5 minutes, and removing the supernatant medium; 6-8mL of fresh growth medium was added, cells were resuspended, and the ratio was 1: 3-1: 10, passaging at 37deg.C, 5% CO ₂ Is cultured in an incubator of (a). Liquid changes were performed every 2-3 days after passaging.

1.1.3. Cell cryopreservation: when the cells grow up to 80-90% of the culture dish, the cells are firstly rinsed by DPBS, and then the DPBS is added again to tap the cell bottle practice report to fall off from the bottle wall; collecting cell suspension into a centrifuge tube, centrifuging at 800 rpm for 5 minutes, and removing the supernatant medium; resuspension of cells with frozen medium, cell counting, and dilution of cells to 2-3×10 ⁶ /mL. Each cryopreservation tube was filled with 1mL of cell cryopreservation suspension. And (3) placing the frozen storage tube of the packaged cells into a frozen storage box, placing the frozen storage box into a refrigerator at the temperature of minus 80 ℃ for overnight storage, and transferring the frozen storage tube into a liquid nitrogen tank.

MDA-MB-231 and MCF 10A cell plating

MDA-MB-231 or MCF 10A cells in the flask were digested with 0.25% pancreatin and suspended in cell culture medium, and added to 96-well plates at 10000 cells per well using a liquid separator for culture at 100. Mu.L per well, 37℃and 5% CO ₂ Culturing overnight.

1.3 screening of polypeptide library

After overnight cell culture, 10. Mu.L of 100. Mu.M polypeptide compound was addedPlacing at 37deg.C, 5% CO ₂ The incubator continues to incubate for 72 hours. On the day of detection, the cells were removed, 10. Mu.L of CCK-8 detection reagent was added, incubated at 37℃for 3 hours, and OD was detected ₄₅₀ Calculating inhibition rate, selecting polypeptide with high inhibition activity, and performing concentration dependency test.

2. Experimental results

By high throughput screening, 2 out of approximately 7.3 ten thousand 80 cyclic peptides were found to be polypeptides SEQ ID NO.1 and SEQ ID NO.2 capable of inhibiting MDA-MB-231 cells, but not inhibiting activity on MCF 10A cells. The inhibitory activity of SEQ ID NO.1 and SEQ ID NO.2 on MDA-MB-231 cells was tested, and the experimental results are shown in Table 2 and FIG. 1.

TABLE 2 concentration response results of SEQ ID NO.1 and SEQ ID NO.1

SEQ ID NO.	MDA-MB-231 cell inhibition IC ₅₀ (μM)	Inhibition of MCF 10A cells by 10. Mu.M
			1	0.51	-13.1％
2	0.74	7.0％

Example 2

Screening of derived peptides

Screening and confirmation of SEQ ID No.1 and SEQ ID No.2 derived polypeptides

The internal decompression technology is applied to decompress the amino acids of the 80 cyclic peptides (SEQ ID NO.1 and SEQ ID NO. 2) to design cyclic peptides or linear peptides with different amino acid sequences of 5-80. Decompressed polypeptides were screened according to the screening procedure in example 1.

2. Experimental results

Decompressing two 80 cyclic peptides of SEQ ID NO.1 and SEQ ID NO.2 to obtain a series of polypeptides with MDA-MB-231 cell inhibition activity, testing the MDA-MB-231 cell inhibition activity, and the experimental results are shown in Table 3 and figures 2-4.

TABLE 3 concentration response results of SEQ ID NO.3 to SEQ ID NO.14

Finally, it should be noted that the above embodiments are only for illustrating the technical solutions of the present application and not for limiting the scope of protection of the present application, and although the present application has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made to the technical solutions of the present application without departing from the spirit and scope of the technical solutions of the present application.

Claims

1. The polypeptide is characterized in that the amino acid sequence of the polypeptide is shown as SEQ ID NO. 1-SEQ ID NO.2 or SEQ ID NO. 3-SEQ ID NO.14, wherein the head and tail amino acids of SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.13 and SEQ ID NO.14 form a ring through peptide bonds; the amino acid sequences SEQ ID NO. 3-SEQ ID NO.14 are linear peptides or cyclic peptides obtained by disassembling the amino acid sequences SEQ ID NO.1 or SEQ ID NO.2.

2. The polypeptide of claim 1, wherein the linear peptide is 22-32 amino acids or 35-45 amino acids in length; the cyclic peptide has a length of 30 to 40 amino acids.

3. The polypeptide of claim 1, wherein the polypeptide amino acid sequence has at least 95% sequence identity to SEQ ID No. 1-SEQ ID No.14 or a derivative of the amino acid sequence shown in modified SEQ ID No. 1-SEQ ID No. 14.

4. A polypeptide according to claim 3, wherein the derivative of the modified amino acid sequence is selected from one or more modifications of: n-terminal and/or C-terminal modification; one or more amino acid residues are substituted with one or more natural and/or unnatural amino acid residues.

5. The polypeptide of claim 4, wherein the modification comprises an amination, a hydroxylation, a carboxylation, a carbonylation, an amidation, an alkylation, a phosphorylation, a glycosylation, a cyclization, a biotinylation, an acetylation, an esterification, a fluorophore modification, a polyethylene glycol PEG modification, an immobilization modification.

6. A polynucleotide molecule comprising one or two polynucleotide molecules capable of encoding the polypeptide of any one of claims 1 to 5.

7. A pharmaceutical composition comprising a therapeutically effective amount of a polypeptide as described above and a pharmaceutically acceptable carrier.

8. Use of a polypeptide molecule according to any one of claims 1 to 5, a polynucleotide molecule according to claim 6 or a pharmaceutical composition according to claim 8 for the preparation of a medicament for the treatment of breast cancer.

9. The use according to claim 8, wherein the breast cancer is a triple negative breast cancer.