Polypeptide bpkr and its application in breast cancer treatment

Abstract

The application discloses a polypeptide BPKR and application thereof in breast cancer treatment, and the polypeptide BPKR has an amino acid sequence of Lys-Thr-Asp-Pro-Val-Leu-Arg-Ser-Leu-Glu-Gly-Leu-Val-Gly-Leu-Gln-Asn-Ile-Arg. The polypeptide BPKR has the activity of inhibiting breast cancer cell proliferation.

Description

Technical Field

[0001] This invention belongs to the field of polypeptide drug technology, specifically relating to polypeptide BPKR and its application in the treatment of breast cancer. Background Technology

[0002] Breast cancer is the most common malignant tumor among women, seriously affecting their physical and mental health and lives. Each year, China accounts for 12.2% of new breast cancer cases and 9.6% of breast cancer deaths worldwide. The treatment of breast cancer has become a major challenge in my country's clinical practice.

[0003] Current treatment methods for breast cancer primarily include surgery, supplemented by chemotherapy, radiotherapy, endocrine therapy, and biological therapy, including molecular targeted therapy. Molecular targeted therapy mainly uses specific monoclonal antibody therapy for breast cancer patients who are positive for HER-2 or other specific proteins. It is generally administered after adjuvant chemotherapy following surgery, but can also be performed concurrently with radiotherapy. However, the number of effective drugs available for breast cancer treatment is limited, and when patients develop drug resistance, it is difficult to find suitable alternatives. Therefore, there is a need to develop more effective drugs for breast cancer treatment.

[0004] Research on peptide-based antitumor drugs has always been highly valued, as they have potential application value in antitumor prodrugs, providing new lead drug prodrugs for targeted cancer therapy. Peptide drugs themselves possess characteristics such as high specificity, good targeting, significant efficacy, and high safety. In the process of antitumor treatment, they can effectively prolong patient survival and improve their quality of life. Therefore, the development of peptide-based antitumor drugs has a promising application prospect for the development of new drugs for cancer treatment. Summary of the Invention

[0005] The purpose of this invention is to provide a polypeptide BPKR and its application in the treatment of breast cancer, which has the activity of inhibiting the proliferation of breast cancer cells.

[0006] The present invention adopts the following technical solution: a polypeptide BPKR, the amino acid sequence of which is: Lys-Thr-Asp-Pro-Val-Leu-Arg-Ser-Leu-Glu-Gly-Leu-Val-Gly-Leu-Gln-Asn-Ile-Arg.

[0007] Furthermore, its molecular formula is C 92 H 163 N 29 O 27 Its molecular weight is 2107.45 g / mol.

[0008] The present invention also discloses derivatives of the polypeptide BPKR, which are obtained by modifying the backbone of the above-mentioned polypeptide BPKR.

[0009] This invention also discloses the application of the above-mentioned polypeptide BPKR in the preparation of breast cancer cell proliferation inhibitors.

[0010] This invention also discloses the application of the above-mentioned polypeptide BPKR in the preparation of MDA-MB-231 and MCF-7 cell proliferation inhibitors.

[0011] The above-mentioned peptide BPKR is used in the preparation of breast cancer cell proliferation inhibitors to inhibit the growth of MDA-MB-231 and / or MCF-7 cells.

[0012] The present invention also discloses a breast cancer treatment drug comprising the above-mentioned polypeptide BPKR, or a derivative thereof comprising the above-mentioned polypeptide BPKR.

[0013] The beneficial effects of this invention are: the polypeptide BPKR has the activity of inhibiting the proliferation of breast cancer cells, providing a new drug candidate and material basis for the treatment of breast cancer. Attached Figure Description

[0014] Figure 1 ESI-MS identification image of peptide BPKR;

[0015] Figure 2 The image shows the RP-HPLC analysis chromatogram of the peptide BPKR.

[0016] Figure 3 This is a graph from an MTT assay showing the antitumor activity of the peptide BPKR.

[0017] 3a shows the time-dependent growth curve of MDA-MB-231 cells;

[0018] 3b shows the time-dependent growth curve of MCF-7 cells;

[0019] Figure 4 Flow cytometry analysis of apoptosis induced by the peptide BPKR in breast cancer cell lines;

[0020] 4a serves as the blank control group for 4b;

[0021] 4b shows the apoptosis-induced effect in MDA-MB-231 cells 48 h after the addition of the peptide BPKR from Example 1;

[0022] 4c is the blank control group for 4d;

[0023] The apoptosis-inducing effect in MCF-7 cells 48 hours after the addition of the peptide BPKR from Example 1 is shown in Figure 4d. Detailed Implementation

[0024] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments.

[0025] This invention discloses a polypeptide BPKR, whose amino acid sequence is: Lys-Thr-Asp-Pro-Val-Leu-Arg-Ser-Leu-Glu-Gly-Leu-Val-Gly-Leu-Gln-Asn-Ile-Arg. Its molecular formula is C1. 92 H 163 N 29 O 27 Its molecular weight is 2107.45 g / mol.

[0026] The present invention also discloses derivatives of the polypeptide BPKR, which are obtained by modifying the backbone of the above-mentioned polypeptide BPKR.

[0027] This invention also discloses the application of the above-mentioned polypeptide BPKR in the preparation of breast cancer cell proliferation inhibitors.

[0028] This invention also discloses the application of the above-mentioned polypeptide BPKR in the preparation of MDA-MB-231 and MCF-7 cell proliferation inhibitors.

[0029] The above-mentioned peptide BPKR is used in the preparation of breast cancer cell proliferation inhibitors to inhibit the growth of MDA-MB-231 and / or MCF-7 cells.

[0030] The present invention also discloses a breast cancer treatment drug comprising the above-mentioned polypeptide BPKR, or a derivative thereof comprising the above-mentioned polypeptide BPKR.

[0031] The abbreviations used in this invention have the following meanings:

[0032] Ala (alanine);

[0033] Arginine (Arginine)

[0034] Asp (aspartic acid);

[0035] Cys: Cysteine;

[0036] Gln (glutamine);

[0037] Glutamic acid;

[0038] His histidine;

[0039] Ile (Isoleucine);

[0040] Glycine;

[0041] Asn asparagine;

[0042] Leucine;

[0043] Lysine;

[0044] Methionine (Met)

[0045] Phenylalanine;

[0046] Proline;

[0047] Serine;

[0048] Threonine;

[0049] Trp tryptophan;

[0050] Tyr (tyrosine);

[0051] Valine;

[0052] Fmoc 9-fluorenyloxycarbonyl;

[0053] MBHA toluene-hydroamine resin;

[0054] DCM (dichloromethane);

[0055] THF tetrahydrofuran;

[0056] DMF N,N-dimethylformamide;

[0057] DIC N,N-diisopropylcarbodiimide;

[0058] HOBt 1-hydroxybenzotriazole;

[0059] DMAP 4-Dimethylaminopyridine;

[0060] PIP (piperidine);

[0061] MeOH (methanol);

[0062] TIS triisopropylsilane;

[0063] RP-HPLC (Reversed-Phase High-Performance Liquid Chromatography)

[0064] TFA (trifluoroacetic acid);

[0065] CH3CN Acetonitrile;

[0066] ESI-MS electrospray ionization mass spectrometry.

[0067] Example 1

[0068] In this embodiment, the BPKR peptide was synthesized using the Fmoc solid-phase synthesis method. The specific synthesis steps are as follows:

[0069] Step 1: Peptide resin coupling:

[0070] 1.5 mmol Rink Amide MBHA resin was suspended in 20 ml DCM for swelling, shaken for 30 min, and then the DCM was removed by vacuum filtration. 2.5 mmol Fmoc-Arg(pbf)-OH was dissolved in 10 ml of a mixture of THF and DMF (11:9 volume ratio), and 3 mmol DIC and 2.5 mmol HOBt were added dropwise. The mixture was stirred in an ice bath for 30 min. The precipitate was then removed by filtration, and the filtrate was added to the swollen resin obtained above. 0.5 mmol DMAP was added, and the reaction was carried out at room temperature for 4 h. The reaction solution was removed by vacuum filtration to obtain Arg-bound resin. A de-Fmoc reagent (4 ml anhydrous pyridine + 4 ml acetic anhydride + 20 ml DMF) was prepared and added to the resin obtained in the above steps, and the reaction was carried out at room temperature for 2 h. After the reaction, the resin was washed sequentially with 10 ml each of DMF, DCM, and MeOH, and then dried under vacuum at room temperature. Add 10 ml of a PIP and DMF mixture (1:4 volume ratio) to the resin, shake to mix, and react at room temperature for 20 min to remove the Fmoc protecting group. Wash the resulting resin three times each with 10 ml of DMF, MeOH, and DCM, and remove the filtrate by vacuum filtration.

[0071] Step 2, peptide chain elongation:

[0072] Dissolve 2.5 mmol Fmoc-Ile-OH and 3 mmol HOBt sequentially in a 10 ml THF and DMF (11:9 volume ratio) mixture, then add 2.5 mmol DIC solution dropwise. React at room temperature for 1 h, filter to remove the precipitate, and add the filtrate to the resin obtained in step one to continue the reaction. Monitor the reaction using the Chloranil method. After the reaction is complete, remove the reaction solution by vacuum filtration. Wash the resin three times with 10 ml DMF, MeOH, and DCM sequentially, and then filter under vacuum.

[0073] Add 5 ml of a PIP and DMF mixture (1:4 volume ratio) to the obtained resin and react at room temperature for 20 min to remove the Fmoc protecting group. Wash the resin three times sequentially with 10 ml of DMF, MeOH, and DCM, and then filter under vacuum. Following the condensation procedure for Fmoc-Ile-OH, sequentially complete the condensation of the remaining 17 amino acids to obtain the 19-peptide-resin complex. Wash the 19-peptide-resin with DCM and dry under vacuum overnight at room temperature.

[0074] Step 3: Peptide resin cutting and purification:

[0075] The 19-peptide-resin obtained in step two was placed in a sintered glass funnel, and 20 ml of freshly prepared cleavage reagent (0.5 ml H₂O + 0.5 ml TIS + 19 ml TFA) was added. The mixture was reacted at room temperature for 2 h. After the reaction was complete, the mixture was vacuum filtered, and the filtrate was concentrated using a rotary evaporator. 100 ml of ice-cold diethyl ether was added to the concentrate, and the precipitate was allowed to form. The precipitate was centrifuged at 5000 rpm for 10 min at 4 °C, and the supernatant was discarded. The precipitate was then vacuum dried to obtain the crude peptide product. The crude peptide was dissolved in water and freeze-dried. The dried crude peptide was dissolved in water, centrifuged at 10000 rpm for 10 min, and the precipitate was removed. The supernatant was then purified using a preparative-grade RP-HPLC system.

[0076] Chromatographic purification conditions: Mobile phase A was an aqueous solution containing 0.1% TFA, mobile phase B was an acetonitrile solution containing 0.1% TFA, and the detection wavelength was set to 220 nm. The elution gradient was 35-55% A for 30 min. The target peak at retention time tR = 14.15 min was collected, and the peptide BPKR was obtained after freeze-drying.

[0077] Example 2

[0078] Mass spectrometry identification and RP-HPLC analysis of peptide BPKR:

[0079] ESI-MS mass spectrometry analysis: The detection method was positive ion detection. The detection result of the peptide BPKR prepared in Example 1 was: [M+2H] 2+ =1054.60, [M+3H] 3+ =707.50, [M+4H] 4+ = 527.95 (the molecular weight of BPKR is 2107.45 g / mol). The mass spectrometry analysis results are consistent with the prediction, confirming the product as the polypeptide BPKR. The results are as follows... Figure 1 As shown.

[0080] RP-HPLC Analysis: The purified BPKR peptide was dissolved in deionized water to a concentration of 1 mg / ml and filtered through a 0.22 μm filter membrane for RP-HPLC analysis. RP-HPLC Analysis Conditions: Mobile phase A was an aqueous solution containing 0.1% TFA; mobile phase B was an acetonitrile solution containing 0.1% TFA; flow rate was 1.0 ml / min; detection wavelength was set to 220 nm. The analytical gradient was set to 35-55% A for 25 min. Figure 2 As shown, the measured retention time t R =11.21 min, purity was 95.04%, the obtained peptide BPKR has high purity, which meets the purity requirements of peptide drugs for subsequent cell biology experiments.

[0081] Example 3

[0082] The polypeptide prepared in Example 1 was subjected to a CCK8 cell proliferation assay:

[0083] Step 1: Culture MDA-MB-231 and MCF-7 cells separately in vitro until they reach 80%–90% confluence, then discard the culture medium. Digest the logarithmic growth phase MDA-MB-231 and MCF-7 cells with trypsin at 5 × 10⁻⁶ ppm. 3 Cells were seeded at a density of 96 wells in an experimental group and a control group. The experimental group was treated with DMEM medium containing 50 μM and 500 μM BPKR, while the control group was treated with DMEM medium containing 10% fetal bovine serum. Each group was divided into 5 replicates and cultured in a constant temperature incubator at 37°C.

[0084] Step 2: After culturing for 24h, 36h, 48h, and 60h, 10μL of CCK8 cell proliferation detection reagent was added to each well, and the cells were cultured in a 37℃ incubator for another 4h. Then, the absorbance of each well was measured at a wavelength of 490nm.

[0085] The measurement results are as follows Figure 3 As shown, 3a is the time-dependent growth curve of MDA-MB-231 cells; 3b is the time-dependent growth curve of MCF-7 cells. The horizontal axis represents treatment time, and the vertical axis represents absorbance values. Absorbance values ​​represent the number of cells and their proliferative capacity; a higher absorbance value indicates a larger number of cells after proliferation, i.e., stronger cell proliferative capacity. Figure 3 It was observed that the proliferation capacity of MDA-MB-231 and MCF-7 cells in the control group continuously increased over time. When the peptide BPKR prepared in Example 1 was added, at a concentration of 50 μM, the proliferation capacity of both MDA-MB-231 and MCF-7 cells showed a decreasing trend over time compared to the control group. At a concentration of 500 μM, the proliferation capacity of both MDA-MB-231 and MCF-7 cells significantly and continuously decreased over time compared to the control group. Specifically, 500 μM BPKR inhibited the proliferation of breast cancer cells MDA-MB-231 and MCF-7 cells, with a 60-hour inhibition rate of 42.26% for MDA-MB-231 cells and 40.37% for MCF-7 cells. This example demonstrates that the peptide BPKR has the ability to inhibit the proliferation of breast cancer cells, effectively slowing down the growth and proliferation of breast cancer cells.

[0086] Example 4

[0087] Step 1: After breast cancer MDA-MB-231 and MCF-7 cells reach the logarithmic growth phase, the cells are digested with trypsin at a concentration of 5 × 10⁻⁶. 5The cells were seeded at a density of / well in 6-well cell culture plates and incubated overnight at 37°C.

[0088] Step 2: The experimental group was treated with DMEM medium containing 500 μM BPKR, while the control group was treated with DMEM medium containing 10% fetal bovine serum. After 48 h, the cells were digested with 0.25% trypsin, centrifuged at 2000 rpm for 5 min to collect the cells, and flow cytometry was used to detect the occurrence of apoptosis in MDA-MB-231 and MCF-7 cells.

[0089] like Figure 4 As shown in the figure, 4a is the blank control group of 4b; Figure 4 b is an apoptosis-inducing diagram of MDA-MB-231 cells 48 h after the addition of the peptide BPKR prepared in Example 1; Figure 4 c represents the blank control group at 4 days; 4 days represents the apoptosis induction plot in MCF-7 cells 48 hours after the addition of the peptide BPKR of this invention; on this two-dimensional scatter plot, the horizontal axis represents cells stained with PI, and the vertical axis represents cells stained with annexin V; among them, cells stained with PI alone are dead cells, cells stained with annexin V alone are cells in the early stage of apoptosis, and cells stained with both are cells in the middle stage of apoptosis. Figure 4 It was found that the apoptosis rates of MDA-MB-231 and MCF-7 cells in the control group were at a low level. When the peptide BPKR prepared in Example 1 was added, and a concentration of 500 μM was used, the apoptosis rates of both MDA-MB-231 and MCF-7 cells increased compared to the control group. Specifically, 500 μM BPKR induced apoptosis in both MDA-MB-231 and MCF-7 breast cancer cells, increasing the apoptosis rate of MDA-MB-231 cells by 17.86% and the apoptosis rate of MCF-7 cells by 19.75%. This example further demonstrates that the peptide BPKR can inhibit breast cancer by inducing apoptosis in breast cancer cells, and has the potential to be used as a drug to inhibit breast cancer.

Claims

1. A polypeptide BPKR, characterized in that, Its amino acid sequence is: KTDPVLRSLEGLVGLQNIR.

2. The use of the polypeptide BPKR as described in claim 1 in the preparation of a drug for treating breast cancer.

3. A breast cancer treatment drug, characterized in that, It contains the polypeptide BPKR as described in claim 1.

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