High-efficiency low-toxicity anti-cancer oligopeptide taking lysine hexanoate as modification unit and application of oligopeptide

Abstract

The invention discloses a high-efficiency low-toxicity anti-cancer oligopeptide taking caproic acid lysine as a modification unit, and belongs to the technical field of biological medicines. The anti-cancer oligopeptide is obtained by respectively modifying caproic acid lysine (K (C6)) at different sites by using LHELLHLLHLLLHH-NH2 as a skeleton structure, the structural general formula of the anti-cancer oligopeptide is Xn-LHELLHLLHLLLHH-NH2, and X represents K (C6); and n represents different modification sites N1, N2, 7 and 14. The anti-cancer oligopeptide is simple and novel in structure and low in manufacturing cost. An in-vitro anti-tumor experiment and a hemolysis experiment show that the anti-cancer oligopeptide has pH-dependent anti-tumor activity on various cancer cells, is low in hemolysis toxicity and shows relatively high selectivity. In addition, scanning electron microscope experiments show that the anti-cancer oligopeptide can quickly kill tumor cells through a pH-dependent membrane rupture mechanism, so that the anti-cancer oligopeptide has considerable application potential in research and development of novel anti-tumor drugs and overcoming of the problem of multidrug resistance. Therefore, the anti-cancer oligopeptide has a good application prospect in the aspect of preparing clinical anti-tumor drugs.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology and relates to a class of high-efficiency, low-toxic anticancer short peptides with hexanoic acid lysine as a modification unit. The present invention also includes the use of such anticancer short peptides in the preparation of clinical anticancer drugs or clinical multidrug-resistant anticancer drugs. Background Art

[0002] Entering the 21st century, with the improvement of human quality of life and the extension of average life expectancy, the pace of life in modern society is accelerating. Coupled with the changes in lifestyle and the influence of multiple factors such as environmental pollution, cancer has surpassed cardiovascular disease and become the primary threat to human life [Int. J. Cancer. 2019, 144 (8): 1941-1953; CA. Cancer J. Clin. 2021, 71 (3): 209-249]. The global statistics released by the World Health Organization's International Agency for Research on Cancer (IARC) in 2022 show that there will be nearly 20 million new cancer cases (including non-melanoma skin cancer) and nearly 10 million cancer deaths (including non-melanoma skin cancer). Based on demographic predictions, by 2050, the number of new cancer cases per year will climb to 35 million, an increase of 77% compared to 2022 [CA. Cancer J. Clin. 2024, 74 (3): 229-263]. There is an urgent need to increase efforts in cancer prevention and treatment to address the evolving and increasing global cancer burden.

[0003] At present, the main treatment strategies for cancer include surgical resection, radiotherapy, chemotherapy, immunotherapy, and molecular targeted therapy [Biomolecules.2021,11(8):1120-1136]. However, the toxic side effects and high recurrence rate of these treatments limit the clinical treatment effect [Biotechnol.Genet.Eng.Rev.2023,39(1):45-84]. Therefore, it is urgent to develop a new anti-tumor drug with high therapeutic ability, low toxicity, high selectivity and low resistance to drug resistance.

[0004] Anticancer peptides (ACPs) are a class of polypeptide molecules with anticancer activity, showing broad-spectrum anticancer properties. They can bind to tumor cell membranes and promote tumor cell death through direct or indirect anticancer mechanisms, thereby exerting anticancer effects [Biomed.Pharmacother.2023,164:114996-115009]. Compared with traditional chemotherapy drugs, anticancer peptides have the advantages of high penetrability, low drug resistance and easy modification, and are more ideal anticancer candidate drugs [Curr.Opin.Pharmacol.2019,47:27-32; Int.J.Mol.Sci.2023,24(16):12931-12965]. However, although anticancer peptides have many advantages over traditional chemotherapy drugs, they also have many defects. For example, their poor stability makes them easy to degrade and become ineffective in the body; their low bioavailability limits their effective effects; their poor selectivity can easily have adverse effects on normal tissues; their high toxicity can cause harm to the body; and they may also induce immune responses during treatment. In addition, factors such as high production costs have limited the widespread application of anticancer peptides in clinical treatment to a certain extent [Int. J. Nanomedicine 2023, 18: 5343-5363].

[0005] Due to hypoxia and abnormal tumor metabolism, the microenvironment around most tumors is more acidic than normal tissues, which is also considered to be an ideal trigger for designing tumor-targeted drugs [Bioconjug.Chem.2018,29(9):2936-2944; J.Control.Release 2021,330:898-906]. The pKa value of the imidazole group contained in histidine is about 6.5, so under acidic conditions when the pH value of the solution is lower than 6.5, the imidazole ring will be protonated and positively charged, while at physiological pH (pH7.2-7.4), histidine is uncharged [J.Drug Target.2021,29(6):651-659; PeerJ.2017,5:e3429-e3429]. Based on this characteristic of histidine, researchers can design acid-sensitive anticancer peptides rich in histidine, which can improve the selectivity of anticancer peptides and reduce toxicity to a certain extent [Asian J.Pharm.Sci.2024,19(1):100890-100901; ACS Omega 2023,8(8):7536-7545]. However, the applicant's research found that this type of acid-sensitive anticancer peptides rich in histidine still needs to be further improved in terms of selectivity improvement and application. Therefore, it is of great research significance to design acid-sensitive anticancer short peptides with high therapeutic index and application potential. Summary of the invention

[0006] One of the purposes of the present invention is to provide a class of anticancer short peptides with simple structure, low manufacturing cost, strong anticancer activity and low toxicity.

[0007] The second purpose of the present invention is to provide the use of the above-mentioned anticancer short peptide in the preparation of clinical anticancer drugs.

[0008] The third object of the present invention is to provide the application of the above-mentioned anticancer short peptide in clinical multidrug-resistant anticancer drugs.

[0009] In order to achieve the above object, the present invention adopts the following technical solutions:

[0010] 1. Structural design of a class of highly effective and low-toxic anticancer short peptides with hexanoic acid lysine as the modification unit

[0011] The present invention provides a class of high-efficiency, low-toxic anticancer short peptides with hexanoic acid lysine as a modification unit, wherein the peptide chain has LHELLHLLHHLLHH-NH2 as a skeleton structure and contains hexanoic acid lysine K (C6) modification units with different modification sites; the general structural formula is X n -LHELLHLLHHLLHH-NH2, where X represents K(C6); n represents different modification sites N 1 、N 2 , 7, 14 digits.

[0012] The amino acid sequences are:

[0013] Lys( N1 C6)-Leu-His-Glu-Leu-Leu-His-Leu-Leu-His-His-Leu-Leu-His-His, recorded as N1 C6-K;

[0014] or:( N2 C6) Lys-Leu-His-Glu-Leu-Leu-His-Leu-Leu-His-His-Leu-Leu-His-His, recorded as N2 C6-K;

[0015] Or: Leu-His-Glu-Leu-Leu-His-Lys(C6)-Leu-His-His-Leu-Leu-His-His, denoted as 7 C6-K;

[0016] Or: Leu-His-Glu-Leu-Leu-His-Leu-Leu-His-His-Leu-Leu-His-Lys(C6), recorded as 14 C6-K.

[0017] As a further preferred embodiment of the technical solution of the present invention, the anticancer short peptide is preferably almost free of hemolytic toxicity. N1 C6-K.

[0018] The above-mentioned anticancer short peptides were all prepared by the classical solid phase synthesis method.

[0019] 2. Application of highly efficient and low-toxic anticancer short peptides with hexanoic acid lysine as the modification unit in the preparation of anticancer drugs

[0020] 1. In vitro anti-tumor experiment

[0021] The anti-tumor activity of the above anti-cancer peptides on different tumor cells was detected by MTT experiment. The selected tumor cell lines include HeLa, MCF-7, A549 and RG cells. The specific operation process is as follows: tumor cells are plated at 1×10 4 / well were seeded in a 96-well plate and cultured overnight at 37°C and 5% CO2. The cells were incubated with serum-free culture medium containing different concentrations of polypeptides (2.5μM, 5μM, 10μM, 20μM, 40μM) at different pH (pH 7.4 / pH 6.0) for 1 hour. Then, 10μL of MTT solution (5mg / mL) was added under light-proof conditions and incubated for another 4 hours. After the incubation, the original culture medium in the well was discarded, and 150μL DMSO was added to dissolve the formazan crystals and oscillated for 10 minutes. Finally, the absorbance at 490nm was measured using an enzyme reader, and the cell survival rate was calculated. The wells treated with blank culture medium at different pH were used as controls. The experiment was repeated three times independently, with four parallel samples each time. The results are as follows Figure 1 shown.

[0022] Figure 1 The results show that the anticancer short peptides of the present invention have obvious pH-dependent antitumor activity against a variety of tumor cells.

[0023] 2. Hemolysis test

[0024] Blood was collected from the eyeballs of healthy Kunming mice (weight 18-22g) in a centrifuge tube containing 200μL of sodium heparin (2mg / mL), centrifuged at 4°C and 800g for 5min, and the supernatant was removed. Then, the cells were washed three times with PBS until the supernatant was clear and the supernatant was discarded again. The red blood cells were then resuspended in PBS to prepare an 8% (v / v) red blood cell suspension. Then, 100μL of red blood cell suspension and 100μL of peptide solutions of different concentrations dissolved in PBS were added to a 96-well plate in sequence and incubated in a 37°C incubator for 1h. After incubation, the 96-well plate was centrifuged at 1200g for 15min. After centrifugation, 100μL of supernatant was aspirated into a new 96-well plate, and the absorbance was finally measured at a wavelength of 490nm using a multifunctional microplate reader. PBS and 2% Triton X-100 were used as negative and positive controls, respectively. The experiment was repeated three times independently, with four parallel samples each time. The results are shown in the figure. Figure 2 shown.

[0025] Figure 2 The results showed that the hemolytic toxicity concentration of the anticancer short peptide of the present invention was higher than the effective concentration of anticancer activity, especially at the N1 position. N1 C6-K has almost no hemolytic toxicity, and its hemolytic toxicity concentration is much higher than the effective concentration of anticancer activity, showing a high therapeutic index and high selectivity.

[0026] 3. Scanning electron microscopy experiment

[0027] MCF-7 cells in logarithmic growth phase were cultured at 2×10 5 The cells were seeded at a density of 100 cells / well on the bottom of a 24-well plate containing a coverslip and incubated overnight. 50 The culture media with different pH values ​​(pH 7.4, pH 6.0) and concentrations of polypeptides were incubated for 1 hour. The cells were then washed twice with PBS, and 1 mL of 2.5% glutaraldehyde was added to each well and fixed overnight at 4°C. The next day, the cells were washed with PBS to remove the glutaraldehyde, and dehydrated with different concentrations of ethanol (50%, 70%, 80%, 90%, 100%) for 15 minutes each, and then dehydrated in tert-butanol for 2 hours. Finally, the samples were dried, sprayed with gold, and observed and imaged using a scanning electron microscope. The cell group treated with blank culture medium (pH 7.4 or pH 6.0) was used as the control group. The results are shown in the figure. Figure 3 shown.

[0028] from Figure 3It can be seen that compared with the control cells without any treatment, the anticancer short peptides of the present invention did not cause obvious changes in cell morphology under the condition of pH 7.4, and the cells after the action of the short peptides still showed a complete cell shape; while under the condition of pH 6.0, the anticancer short peptides can quickly destroy the tumor cell membrane and kill tumor cells, indicating an obvious pH-dependent membrane disruption mechanism. This unique membrane disruption mechanism gives it a significant advantage in combating multidrug resistance.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] 1. The present invention uses the histidine-rich acid-sensitive anticancer peptide sequence LHELLHLLHHLLHH-NH2 as the skeleton structure, introduces hexanoic acid lysine modification units at different sites, and obtains a class of high-efficiency and low-toxic anticancer short peptides; the anticancer short peptide has a simple structure, low molecular weight, and novel design. It can be successfully prepared by conventional solid-phase synthesis. This synthesis method greatly reduces the production cost, has good economy and operability, and lays a solid foundation for its large-scale production and wide application.

[0031] 2. In vitro anti-tumor experiments and hemolytic experiments showed that this type of anti-cancer short peptides have pH-dependent anti-tumor activity and low hemolytic toxicity against a variety of cancer cells, with a high therapeutic index and high selectivity. In addition, scanning electron microscopy experiments showed that this type of anti-cancer short peptides can quickly kill tumor cells through a pH-dependent membrane-breaking mechanism, making it have considerable application potential in the development of new anti-cancer drugs and overcoming the problem of multidrug resistance. Therefore, this type of anti-cancer short peptide has good application prospects in the preparation of clinical anti-cancer drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 The in vitro anti-tumor activity test diagram of the anti-cancer short peptide of the present invention on HeLa, MCF-7, A549 and RG cells;

[0033] Figure 2 This is a diagram of the hemolysis experiment of the anticancer short peptide of the present invention;

[0034] Figure 3 This is a scanning electron microscope experimental picture of the anticancer short peptide of the present invention;

[0035] Figure 4 The anticancer short peptide of the present invention N1 Mass spectrum of C6-K;

[0036] Figure 5 The anticancer short peptide of the present invention N2 Mass spectrum of C6-K;

[0037] Figure 6 The anticancer short peptide of the present invention 7 Mass spectrum of C6-K;

[0038] Figure 7 The anticancer short peptide of the present invention 14 Mass spectrum of C6-K. DETAILED DESCRIPTION

[0039] The synthesis process of the anticancer short peptide of the present invention is further described below through specific examples.

[0040] Embodiment 1: N1 Synthesis of C6-K

[0041] 1) Resin activation and pretreatment

[0042] Weigh 0.543 g (0.25 mmol) of MBHA resin into a synthesizer, and add 15 mL of DCM to swell the resin for 30 min. After swelling, wash the resin three times with DMF, and then use the ninhydrin colorimetric method to test the resin. If the resin is colorless and transparent, it indicates that the resin is normal and usable.

[0043] 2) N1 Synthesis of C6-K-resin

[0044] Add a DMF solution (v / v) containing 20% ​​piperidine to the above-mentioned normal resin and use the ninhydrin colorimetric method to detect whether the Fmoc protecting group of the resin has been removed. If the resin is bluish purple, it means that the Fmoc protecting group has been removed. Then wash the resin 4 times with DMF. Then weigh Fmoc-His(Trt)-OH (0.75mmol), HBTU (0.75mmol), HOBT (0.75mmol) and DIEA (1.5mmol), dissolve them in DMF, add them to the synthesizer, and stir to react for 1h. After the reaction is completed, the resin is colorless and transparent by the ninhydrin colorimetric method, and Fmoc-His(Trt)-resin is obtained.

[0045] According to the above method, the subsequent amino acid synthesis was carried out through condensation reactions to obtain Fmoc-Lys(Dde)-Leu-His(Trt)-Glu(OtBu)-Leu-Leu-His(Trt)-Leu-Leu-His(Trt)-His(Trt)-Leu-Leu-His(Trt)-His(Trt)-resin. Then, the Dde protecting group of the N-terminal lysine side chain was removed with a DMF solution containing 2% hydrazine hydrate, and CH3(CH2)4COOH (0.75 mmol), HBTU (0.75 mmol), HOBT (0.75 mmol) and DIEA (1.5 mmol) were added for condensation reaction for 1 hour. After the reaction was completed, the resin was colorless when detected by the ninhydrin colorimetric method, and the obtained N1 C6-K-resin

[0046] 3) Peptide cleavage

[0047] The obtained KLHE-resin was washed with DCM and MeOH in turn, the resin was compressed and dried until it became powdery, and then the cutting agent TFA: Tis: H2O = 9.5: 0.25: 0.25 (v: v: v) was added to react for 3 h to remove the resin and all side chain protecting groups. The cutting solution was collected and trifluoroacetic acid was removed using a rotary evaporator, and then extracted with deionized water and ice ether, and the extract was freeze-dried to obtain the crude peptide.

[0048] 4) Peptide purification

[0049] The crude peptide sample obtained above was separated and purified by RP-HPLC, and then freeze-dried to obtain pure peptide. N1 C6-K. The molecular weight was 1987.18Da as determined by mass spectrometry. Figure 4 .

[0050] Embodiment 2: N2 Synthesis of C6-K

[0051] 1) Resin activation and pretreatment

[0052] Same as Example 1.

[0053] 2) N2 Synthesis of C6-K-resin

[0054] Add a DMF solution (v / v) containing 20% ​​piperidine to the above-mentioned normal resin and use the ninhydrin colorimetric method to detect whether the Fmoc protecting group of the resin has been removed. If the resin is bluish purple, it means that the Fmoc protecting group has been removed. Then wash the resin with DMF 4 times. Then weigh Fmoc-His(Trt)-OH (0.75mmol), HBTU (0.75mmol), HOBT (0.75mmol) and DIEA (1.5mmol), dissolve them in DMF, add them to the synthesizer, and stir to react for 1h. After the reaction is completed, the resin is colorless and transparent by the ninhydrin colorimetric method to obtain Fmoc-His(Trt)-resin.

[0055] According to the above method, the subsequent amino acid synthesis was carried out through condensation reactions to obtain Fmoc-Lys(Boc)-Leu-His(Trt)-Glu(OtBu)-Leu-Leu-His(Trt)-Leu-Leu-His(Trt)-His(Trt)-Leu-Leu-His(Trt)-His(Trt)-resin. Then, the Fmoc group at the end of the peptide chain was removed with a DMF solution containing 20% ​​piperidine, and CH3(CH2)4COOH (0.75mmol), HBTU (0.75mmol), HOBT (0.75mmol) and DIEA (1.5mmol) were added for condensation reaction for 1h. After the reaction was completed, the resin was colorless when detected by the ninhydrin colorimetric method, and the obtained N2 C6-K-resin.

[0056] 3) Peptide cleavage

[0057] Same as Example 1.

[0058] 4) Peptide purification

[0059] Same as Example 1, molecular weight is 1987.13Da, mass spectrum is as follows Figure 5 shown.

[0060] Embodiment 3: 7 Synthesis of C6-K

[0061] 1) Resin activation and pretreatment

[0062] Same as Example 1.

[0063] 2) 7 C6-K-resin synthesis

[0064] Add a DMF solution (v / v) containing 20% ​​piperidine to the above-mentioned normal resin and use the ninhydrin colorimetric method to detect whether the Fmoc protecting group of the resin has been removed. If the resin is bluish purple, it means that the Fmoc protecting group has been removed. Then wash the resin with DMF 4 times. Then weigh Fmoc-His(Trt)-OH (0.75mmol), HBTU (0.75mmol), HOBT (0.75mmol) and DIEA (1.5mmol), dissolve them in DMF, add them to the synthesizer, and stir to react for 1h. After the reaction is completed, the resin is colorless and transparent by the ninhydrin colorimetric method to obtain Fmoc-His(Trt)-resin.

[0065] According to the above method, the subsequent amino acid synthesis was carried out by condensation reaction in sequence to obtain Fmoc-Leu-His(Trt)-Glu(OtBu)-Leu-Leu-His(Trt)-Lys(Dde)-Leu-His(Trt)-His(Trt)-Le u-Leu-His(Trt)-His(Trt)-resin. Then, the side chain protecting group Dde of Lys(Dde) was removed with a DMF solution containing 2% hydrazine hydrate. CH3(CH2)4COOH (0.75mmol), HBTU (0.75mmol), HOBT (0.75mmol) and DIEA (1.5mmol) were added to carry out the condensation reaction for 1h. After the reaction was completed, the resin was colorless when detected by the ninhydrin colorimetric method, and the obtained 7 C6-K-esin.

[0066] 3) Peptide cleavage

[0067] Same as Example 1.

[0068] 4) Peptide purification

[0069] Same as Example 1, molecular weight is 1874.8Da, mass spectrum is as follows Figure 6 shown.

[0070] Embodiment 4: 14 Synthesis of C6-K

[0071] 1) Resin activation and pretreatment

[0072] Same as Example 1.

[0073] 2) 14 Synthesis of C6-K-resin

[0074] Add a DMF solution (v / v) containing 20% ​​piperidine to the above-mentioned normal resin and use the ninhydrin colorimetric method to detect whether the Fmoc protecting group of the resin has been removed. If the resin is bluish purple, it means that the Fmoc protecting group has been removed. Then wash the resin with DMF 4 times. Then weigh Fmoc-Lys(Dde)-OH (0.75mmol), HBTU (0.75mmol), HOBT (0.75mmol) and DIEA (1.5mmol), dissolve them in DMF, add them to the synthesizer, and stir to react for 1h. After the reaction is completed, the resin is colorless and transparent by the ninhydrin colorimetric method to obtain Fmoc-Lys(Dde)-resin.

[0075] According to the above method, the subsequent amino acid synthesis is carried out through condensation reactions to obtain Fmoc-Leu-His(Trt)-Glu(OtBu)-Leu-Leu-His(Trt)-Leu-Leu-His(Trt)-His(Trt)-Leu-Leu-His(Trt)-Lys(Dde)-resin. Then, the side chain protecting group Dde of the C-terminal lysine is removed with a DMF solution containing 2% hydrazine hydrate. CH3(CH2)4COOH (0.75mmol), HBTU (0.75mmol), HOBT (0.75mmol) and DIEA (1.5mmol) are added to carry out the condensation reaction for 1h. After the reaction is completed, the resin is colorless when detected by the ninhydrin colorimetric method, and the obtained 14 C6-K-resin.

[0076] 3) Peptide cleavage

[0077] Same as Example 1.

[0078] 4) Peptide purification

[0079] Same as Example 1, molecular weight is 1850.07Da, mass spectrum is as follows Figure 7 shown.

Claims

1. A class of highly effective and low-toxic anticancer short peptides with hexanoic acid lysine as a modification unit, characterized in that: The anticancer short peptide chain has LHELLHLLHHLLHH-NH2 as the skeleton structure and contains hexanoic acid lysine K (C6) modification units with different modification sites; the amino acid sequences are: Lys( N1 C6)-Leu-His-Glu-Leu-Leu-His-Leu-Leu-His-His-Leu-Leu-His-His, denoted as N1 C6-K; Or: ( N2 C6) Lys-Leu-His-Glu-Leu-Leu-His-Leu-Leu-His-His-Leu-Leu-His-His, denoted as N2 C6-K; Or: Leu-His-Glu-Leu-Leu-His-Lys(C6)-Leu-His-His-Leu-Leu-His-His, denoted as 7 C6-K; Or: Leu-His-Glu-Leu-Leu-His-Leu-Leu-His-Leu-Leu-His-His-Leu-Leu-His-Lys(C6), refer to 14 C6-K.

2. A class of high-efficiency, low-toxic anticancer short peptides with hexanoic acid lysine as a modification unit as claimed in claim 1, characterized in that: The anticancer short peptide is N1 C6-K.

3. Use of a class of high-efficiency, low-toxic anticancer short peptides with hexanoic acid lysine as a modification unit as described in claim 1 or 2 in the preparation of clinical anticancer drugs.

4. The use of a class of high-efficiency and low-toxic anticancer short peptides with hexanoic acid lysine as a modification unit in the preparation of clinical anticancer drugs as claimed in claim 3, characterized in that: The anticancer drug is used to treat cervical cancer, breast cancer, lung cancer or liver cancer.

5. Use of a class of high-efficiency, low-toxic anticancer short peptides with hexanoic acid lysine as a modification unit as claimed in claim 3 in the preparation of clinical multidrug-resistant anticancer drugs.

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