Highly efficient and low-toxicity anticancer short peptides with lysine caproic acid as modification unit and application thereof

Abstract

The application discloses a kind of efficient low-toxicity anticancer short peptide with hexanoic acid lysine as modification unit, and belongs to the technical field of biological medicine.The anticancer short peptide is with LHELLHLLHHLLHH-NH2 as skeleton structure, hexanoic acid lysine (K(C6)) is modified at different sites to obtain, and its structural general formula is X n -LHELLHLLHHLLHH-NH2, wherein X represents K(C6);n represents different modification sites N 1 、N 2 、7、14 position.The anticancer short peptide has simple structure, novel, low manufacturing cost.In vitro antitumor experiment, hemolysis experiment show that the anticancer short peptide has pH-dependent antitumor activity, low hemolytic toxicity to a variety of cancer cells, and shows high selectivity.In addition, scanning electron microscopy experiment shows that the anticancer short peptide can quickly kill tumor cells through pH-dependent membrane rupture mechanism, so that it has considerable application potential in the development of new antitumor drugs and overcoming the problem of multi-drug resistance.Therefore, the anticancer short peptide has good application prospect in the preparation of clinical antitumor drugs.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to a class of highly efficient and low-toxicity anticancer short peptides modified with hexanoic acid lysine. This invention also includes the application of such anticancer short peptides in the preparation of clinical anticancer drugs or clinical multidrug-resistant anticancer drugs. Background Technology

[0002] Entering the 21st century, with the improvement of human living standards and the extension of average lifespan, the pace of modern life has been accelerating. Coupled with the changes in lifestyle and the influence of multiple factors such as environmental pollution, cancer has surpassed cardiovascular disease to become the primary threat to human life [Int.J.Cancer.2019,144(8):1941-1953; CA.CancerJ.Clin.2021,71(3):209-249]. According to the 2022 global statistics released by the International Agency for Research on Cancer (IARC) of the World Health Organization, there were 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 projections, by 2050, the number of new cancer cases per year will climb to 35 million, an increase of 77% compared to 2022 [CA.CancerJ.Clin.2024,74(3):229-263]. Therefore, there is an urgent need to increase efforts in cancer prevention and treatment to address the growing global cancer burden.

[0003] Currently, major cancer treatment strategies encompass a variety of approaches, including surgical resection, radiotherapy, chemotherapy, immunotherapy, and molecular targeted therapy [Biomolecules. 2021, 11(8): 1120-1136]. However, the toxic side effects and high recurrence rates of these treatments limit their clinical efficacy [Biotechnol. Genet. Eng. Rev. 2023, 39(1): 45-84]. Therefore, the development of novel antitumor drugs with high therapeutic efficacy, low toxicity, high selectivity, and low likelihood of inducing drug resistance is urgently needed.

[0004] Anticancer peptides (ACPs) are a class of polypeptide molecules with anticancer activity, exhibiting broad-spectrum anticancer properties. They can bind to tumor cell membranes and induce 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 advantages such as high penetration, low resistance to drug resistance, and ease of modification, making them ideal candidate drugs for anticancer treatment [Curr. Opin. Pharmacol. 2019, 47: 27-32; Int. J. Mol. Sci. 2023, 24(16): 12931-12965]. However, despite the many advantages of anticancer peptides compared to traditional chemotherapy drugs, they also have many drawbacks. For example, their poor stability leads to easy degradation and inactivation in vivo; low bioavailability limits their effectiveness; poor selectivity can easily have adverse effects on normal tissues; high toxicity can harm the body; and they may also trigger immune responses during treatment. In addition, the high production cost also limits the widespread application of anticancer peptides in clinical treatment to some extent [Int.J.Nanomedicine 2023,18:5343-5363].

[0005] Due to hypoxia and abnormal tumor metabolic processes, the microenvironment surrounding most tumors is more acidic than that of normal tissues, which is also considered an ideal trigger for designing targeted tumors [Bioconjug. Chem. 2018, 29(9): 2936-2944; J. Control. Release 2021, 330: 898-906]. The pKa value of the imidazole group in histidine is about 6.5. Therefore, under acidic conditions, when the solution pH is below 6.5, the imidazole ring will be protonated and positively charged, while at physiological pH (pH 7.2-7.4), histidine is uncharged [J. Drug Target. 2021, 29(6): 651-659; Peer J. 2017, 5: e3429-e3429]. Based on the characteristic of histidine, researchers can design acid-sensitive anticancer peptides rich in histidine, which can improve the selectivity and reduce the toxicity of anticancer peptides 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 peptide rich in histidine still needs further improvement in terms of selectivity and application. Therefore, designing acid-sensitive short anticancer peptides with high therapeutic index and application potential is of great research significance. Summary of the Invention

[0006] One of the objectives of this invention is to provide a class of anticancer short peptides that are simple in structure, low in manufacturing cost, have strong anticancer activity, and low toxicity.

[0007] The second objective of this invention is to provide the application of the above-mentioned anticancer short peptides in the preparation of clinical anticancer drugs.

[0008] The third objective of this invention is to provide the application of the above-mentioned anticancer short peptides in clinical multidrug-resistant anticancer drugs.

[0009] To achieve the above objectives, the present invention adopts the following technical solution:

[0010] I. Structural Design of a Class of Highly Efficient and Low-Toxicity Anticancer Short Peptides Modified with Hexanoic Acid and Lysine

[0011] This invention provides a class of highly efficient and low-toxicity anticancer short peptides modified with hexanoic acid lysine as the modification unit. The peptide chain has an LHELLHLLHHLLHH-NH2 backbone structure and contains hexanoic acid lysine K(C6) modification units at different modification sites; its general structural formula is X. n -LHELLHLLHHLLHH-NH2, where X represents K(C6); n represents different modification sites N 1 N 2 7, 14.

[0012] Its amino acid sequences are as follows:

[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 one with almost no hemolytic toxicity. N1 C6-K.

[0018] All of the above-mentioned anticancer short peptides were prepared using the classic solid-phase synthesis method.

[0019] II. Application of Highly Efficient and Low-Toxicity Anticancer Short Peptides Modified with Hexanoic Acid Lysine in the Preparation of Anticancer Drugs

[0020] 1. In vitro antitumor experiment

[0021] The antitumor activity of the above-mentioned anticancer short peptides against different tumor cells was detected by MTT assay. The selected tumor cell lines included HeLa, MCF-7, A549, and RG cells. The specific procedure was as follows: tumor cells were inoculated at 1×10⁻⁶ cells / cells. 4 Cells were seeded at a density of 1 / 2 well in 96-well plates and cultured overnight at 37°C and 5% CO2. Cells were then co-incubated for 1 h with serum-free medium containing different concentrations of peptides (2.5 μM, 5 μM, 10 μM, 20 μM, 40 μM) at different pH values ​​(pH 7.4 / pH 6.0). Then, 10 μL of MTT solution (5 mg / mL) was added under dark conditions, and incubation continued for 4 h. After incubation, the original culture medium in the wells was discarded, and 150 μL of LDMSO was added to dissolve formazan crystals. The mixture was shaken for 10 min. Finally, the absorbance at 490 nm was measured using a microplate reader, and cell viability was calculated. Wells treated with blank medium at different pH values ​​served as controls. The experiment was independently repeated three times, with four replicates per experiment. Results are as follows: Figure 1 As shown.

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

[0023] 2. Hemolysis test

[0024] Blood was collected from the eyes of healthy Kunming mice (18-22g) in centrifuge tubes containing 200μL of heparin sodium (2mg / mL). The tubes were centrifuged at 800g for 5 min at 4℃, and the supernatant was removed. The cells were then washed three times with PBS until the supernatant was clear, and this supernatant was discarded again. The red blood cells were then resuspended in PBS to prepare an 8% (v / v) red blood cell suspension. 100μL of the red blood cell suspension and 100μL of peptide solutions of different concentrations dissolved in PBS were added sequentially to 96-well plates, and the plates were incubated at 37℃ for 1 h. After incubation, the 96-well plates were centrifuged at 1200g for 15 min. After centrifugation, 100μL of the supernatant was transferred to a new 96-well plate, and the absorbance was measured at 490nm using a multi-mode microplate reader. PBS and 2% Triton X-100 were used as negative and positive controls, respectively. The experiment was independently repeated three times, with four replicates per experiment. The results are as follows: Figure 2 As shown.

[0025] Figure 2 The results showed that the hemolytic toxicity concentrations of the anticancer short peptides of this invention were all higher than the effective concentrations for anticancer activity, especially at the N1 position. The short peptide analogs we obtained using K(C6) modification... N1 C6-K has almost no hemolytic toxicity, and its hemolytic toxicity concentration is much higher than the effective concentration for 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 fed at a rate of 2 × 10⁻⁶. 5 Cells were seeded at a density of 1 cell / well at the bottom of 24-well plates containing coverslips and incubated overnight. Subsequently, the cells were washed with PBS and incubated with IC50-containing... 50 Cells were co-incubated for 1 hour in mediums with different concentrations of peptide at different pH values ​​(pH 7.4, pH 6.0). Afterwards, the cells were washed twice with PBS, and 1 mL of 2.5% glutaraldehyde was added to each well for fixation overnight at 4°C. The next day, the cells were washed with PBS to remove glutaraldehyde, and then dehydrated with different concentrations of ethanol (50%, 70%, 80%, 90%, 100%) for 15 minutes each time, followed by dehydration in tert-butanol for 2 hours. Finally, the samples were dried, sputter-coated with gold, and observed and imaged using a scanning electron microscope. Cells treated with blank medium (pH 7.4 or pH 6.0) served as a control group. Results are as follows: Figure 3 As shown.

[0028] from Figure 3As can be seen, compared with untreated control cells, the anticancer short peptides of the present invention did not cause significant changes in cell morphology at pH 7.4, and the cells treated with the short peptides still exhibited intact cell shape; while at pH 6.0, these anticancer short peptides could rapidly destroy tumor cell membranes and kill tumor cells, demonstrating a clear pH-dependent membrane-breaking mechanism. This unique membrane-breaking mechanism gives it a significant advantage in combating multidrug resistance.

[0029] The advantages of this invention compared to the prior art are as follows:

[0030] 1. This invention uses the histidine-rich acid-sensitive anticancer peptide sequence LHELLHLLHHLLHH-NH2 as the backbone structure, and introduces hexanoic acid lysine modification units at different sites to obtain a class of highly efficient and low-toxicity anticancer short peptides. These anticancer short peptides have a simple structure, low molecular weight, and novel design. They can be successfully prepared using conventional solid-phase synthesis methods. This synthesis method greatly reduces production costs and has good economic efficiency and operability, laying a solid foundation for their large-scale production and widespread application.

[0031] 2. In vitro antitumor experiments and hemolysis experiments showed that these anticancer short peptides exhibit pH-dependent antitumor activity and low hemolytic toxicity against various cancer cells, with a high therapeutic index and high selectivity. Furthermore, scanning electron microscopy experiments demonstrated that these anticancer short peptides can rapidly kill tumor cells through a pH-dependent membrane-permeability mechanism, giving them considerable application potential in the development of novel anticancer drugs and in overcoming multidrug resistance. Therefore, these anticancer short peptides show promising application prospects in the preparation of clinical anticancer drugs. Attached Figure Description

[0032] Figure 1 This is a diagram illustrating the in vitro antitumor activity of the anticancer short peptide of this invention against HeLa, MCF-7, A549, and RG cells.

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

[0034] Figure 3 This is a scanning electron microscope image of the anticancer short peptide of this invention;

[0035] Figure 4 This invention relates to an anticancer short peptide. N1 Mass spectrum of C6-K;

[0036] Figure 5 This invention relates to an anticancer short peptide. N2 Mass spectrum of C6-K;

[0037] Figure 6 This invention relates to an anticancer short peptide. 7 Mass spectrum of C6-K;

[0038] Figure 7 This invention relates to an anticancer short peptide. 14 Mass spectrum of C6-K. Detailed Implementation

[0039] The synthesis process of the anticancer short peptide of the present invention will be further explained below through specific embodiments.

[0040] Example 1: N1 C6-K Synthesis

[0041] 1) Resin activation and pretreatment

[0042] Weigh 0.543 g (0.25 mmol) of MBHA resin into the synthesizer and add 15 mL of DCM to swell the resin for 30 min. After swelling is complete, wash the resin three times with DMF. 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 resin that has passed the test, and test whether the Fmoc protecting group of the resin has been removed by the ninhydrin colorimetric method. If the resin turns blue-purple, it indicates that the Fmoc protecting group has been removed. Then wash the resin four times with DMF. Next, weigh out Fmoc-His(Trt)-OH (0.75 mmol), HBTU (0.75 mmol), HOBT (0.75 mmol), and DIEA (1.5 mmol), dissolve them in DMF, and add them to the synthesizer. Stir the reaction for 1 hour. After the reaction is complete, the resin is colorless and transparent as determined by the ninhydrin colorimetric method, yielding Fmoc-His(Trt)-resin.

[0045] Following the above method, subsequent amino acid synthesis via condensation reactions yielded 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. The Dde protecting group on the N-terminal lysine side chain was then removed using a DMF solution containing 2% hydrazine hydrate. CH3(CH2)4COOH (0.75 mmol), HBTU (0.75 mmol), HOBT (0.75 mmol), and DIEA (1.5 mmol) were then added, and the condensation reaction proceeded for 1 h. After the reaction was complete, the resin was colorless as determined by the ninhydrin colorimetric method, yielding... N1 C6-K-resin

[0046] 3) Peptide cleavage

[0047] The obtained KLHE-resin was washed sequentially with DCM and MeOH. The resin was compressed and dried until it became powder. Then, a cleavage agent TFA:Tis:H2O = 9.5:0.25:0.25 (v:v:v) was added, and the mixture was reacted for 3 hours to remove the resin and all side-chain protecting groups. The cleavage solution was collected, and trifluoroacetic acid was removed using a rotary evaporator. The solution was then extracted with deionized water and ice-cold diethyl ether. 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 determined by mass spectrometry to be 1987.18 Da. The mass spectrum is shown below. Figure 4 .

[0050] Example 2: N2 C6-K Synthesis

[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 resin that has passed the test, and test whether the Fmoc protecting group of the resin has been removed by the ninhydrin colorimetric method. If the resin turns blue-purple, it indicates that the Fmoc protecting group has been removed. Then wash the resin four times with DMF. Next, weigh out Fmoc-His(Trt)-OH (0.75 mmol), HBTU (0.75 mmol), HOBT (0.75 mmol), and DIEA (1.5 mmol), dissolve them in DMF, and add them to the synthesizer. Stir the reaction for 1 hour. After the reaction is complete, the resin is colorless and transparent as determined by the ninhydrin colorimetric method, yielding Fmoc-His(Trt)-resin.

[0055] Following the above method, subsequent amino acid synthesis via condensation reactions yielded 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. The Fmoc groups at the peptide ends were then removed using a DMF solution containing 20% ​​piperidine. CH3(CH2)4COOH (0.75 mmol), HBTU (0.75 mmol), HOBT (0.75 mmol), and DIEA (1.5 mmol) were then added, and the condensation reaction proceeded for 1 h. After the reaction was complete, the resin was colorless as determined by the ninhydrin colorimetric method, yielding... N2 C6-K-resin.

[0056] 3) Peptide cleavage

[0057] Same as Example 1.

[0058] 4) Peptide purification

[0059] Same as Example 1, with a molecular weight of 1987.13 Da, and the mass spectrum is as follows. Figure 5 As shown.

[0060] Example 3: 7 C6-K Synthesis

[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 resin that has passed the test, and test whether the Fmoc protecting group of the resin has been removed by the ninhydrin colorimetric method. If the resin turns blue-purple, it indicates that the Fmoc protecting group has been removed. Then wash the resin four times with DMF. Next, weigh out Fmoc-His(Trt)-OH (0.75 mmol), HBTU (0.75 mmol), HOBT (0.75 mmol), and DIEA (1.5 mmol), dissolve them in DMF, and add them to the synthesizer. Stir the reaction for 1 hour. After the reaction is complete, the resin is colorless and transparent as determined by the ninhydrin colorimetric method, yielding Fmoc-His(Trt)-resin.

[0065] Following the above method, subsequent amino acid synthesis via condensation reactions yielded Fmoc-Leu-His(Trt)-Glu(OtBu)-Leu-Leu-His(Trt)-Lys(Dde)-Leu-His(Trt)-His(Trt)-Leu-Leu-His(Trt)-His(Trt)-resin. The protecting group Dde of Lys(Dde) was then removed using a DMF solution containing 2% hydrazine hydrate. CH3(CH2)4COOH (0.75 mmol), HBTU (0.75 mmol), HOBT (0.75 mmol), and DIEA (1.5 mmol) were then added, and the condensation reaction proceeded for 1 h. After the reaction was complete, the resin was colorless as determined by the ninhydrin colorimetric method, yielding... 7 C6-K-esin.

[0066] 3) Peptide cleavage

[0067] Same as Example 1.

[0068] 4) Peptide purification

[0069] Same as Example 1, with a molecular weight of 1874.8 Da, and the mass spectrum is as follows. Figure 6 As shown.

[0070] Example 4: 14 C6-K Synthesis

[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 resin that has passed the test, and test whether the Fmoc protecting group of the resin has been removed by the ninhydrin colorimetric method. If the resin turns blue-purple, it indicates that the Fmoc protecting group has been removed. Then wash the resin four times with DMF. Next, weigh out Fmoc-Lys(Dde)-OH (0.75 mmol), HBTU (0.75 mmol), HOBT (0.75 mmol), and DIEA (1.5 mmol), dissolve them in DMF, and add them to the synthesizer. Stir and react for 1 hour. After the reaction is complete, the resin is colorless and transparent as determined by the ninhydrin colorimetric method, yielding Fmoc-Lys(Dde)-resin.

[0075] Following the above method, subsequent amino acid synthesis via condensation reactions yielded Fmoc-Leu-His(Trt)-Glu(OtBu)-Leu-Leu-His(Trt)-Leu-Leu-His(Trt)-His(Trt)-Leu-Leu-His(Trt)-Lys(Dde)-resin. The C-terminal lysine side-chain protecting group Dde was then removed using a DMF solution containing 2% hydrazine hydrate. CH3(CH2)4COOH (0.75 mmol), HBTU (0.75 mmol), HOBT (0.75 mmol), and DIEA (1.5 mmol) were then added, and the condensation reaction proceeded for 1 h. After the reaction was complete, the resin was colorless as determined by the ninhydrin colorimetric method, yielding... 14 C6-K-resin.

[0076] 3) Peptide cleavage

[0077] Same as Example 1.

[0078] 4) Peptide purification

[0079] Same as Example 1, with a molecular weight of 1850.07 Da, and the mass spectrum is as follows. Figure 7 As shown.

Claims

1. A class of highly efficient, low-toxicity anticancer short peptides modified with hexanoic acid lysine, characterized in that, The anticancer short peptide chain has an LHELLHLLHHLLHH-NH2 backbone structure and contains hexanoic acid lysine K(C6) modification units at different modification sites; its amino acid sequences are as follows: Lys( N1 C6)-Leu-His-Glu-Leu-Leu-His-Leu-Leu-His-Leu-Leu-His-His-Leu-Leu-His-His, referred to as N1 C6-K; or: ( N2 C6)Lys-Leu-His-Glu-Leu-Leu-His-Leu-Leu-His-His-Leu-Leu-His-His, designated 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. The highly efficient and low-toxicity anticancer short peptide with hexanoic acid lysine as a modification unit as described in claim 1, characterized in that, The anticancer short peptide is N1 C6-K.

3. The application of a class of highly efficient and low-toxicity anticancer short peptides modified with hexanoic acid lysine as a unit as described in claim 1 or 2 in the preparation of clinical anticancer drugs, characterized in that, The cancer mentioned is cervical cancer, breast cancer, lung cancer, or liver cancer.

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