Nitrogen-terminal fatty acid modified anticancer peptide and application thereof

Abstract

The invention belongs to the field of polypeptides, and particularly relates to a nitrogen-terminal fatty acid modified anticancer peptide and application thereof. The nitrogen terminal of the anti-cancer peptide is modified by straight-chain fatty acid, the carbon terminal of the anti-cancer peptide is of an amide structure, and the structural general formula of the anti-cancer peptide is Cn-KaWbEc, wherein Cn represents a fatty acid chain with n carbon atoms, a is the number of lysine K, b is the number of tryptophan W, and c is the number of glutamic acid E; n is equal to 6-18; a + b + c = 8; a = 3 or 4, b = 3 or 4, and c = 0, 1 or 2. According to the anti-cancer peptide modified by the nitrogen-terminal fatty acid, lysine, tryptophan and glutamic acid are used for constructing 8-bit oligopeptide, a nitrogen-terminal fatty acid modification strategy and a carbon-terminal amide structure are matched, and the anti-cancer peptide is simple in structure and low in synthesis cost. In-vitro anti-tumor experiments and toxicity experiments show that the anti-cancer peptide has remarkable anti-tumor activity on various tumor cells such as 4T1, Hela, A549, HCCLM3 and the like, and shows broad-spectrum anti-cancer activity.

Description

Technical Field

[0001] This invention belongs to the field of polypeptides, specifically relating to a class of anticancer peptides modified with nitrogen-terminal fatty acids and their applications. Background Technology

[0002] Cancer has long threatened human health and is one of the leading causes of death worldwide. Chemotherapy has played a crucial role in cancer treatment; however, traditional chemotherapy drugs suffer from poor selectivity, significant toxic side effects, and a tendency to induce drug resistance in tumor cells. Therefore, the development of novel anticancer drugs is of great importance for cancer treatment.

[0003] Anticancer peptides are widely found in animals, plants, and microorganisms, and possess broad-spectrum anticancer activity. In addition, there are a large number of artificially synthesized anticancer peptides. Mini-Rev. Med. Chem [2021, 21, 58-68]. Most anticancer peptides can selectively act on the negatively charged tumor cell membrane surface through their positive charge, thereby killing tumor cells through various mechanisms such as membrane lysis, induction of apoptosis, or inhibition of angiogenesis. Molecules, [2023, 28, 1148] Compared to traditional chemotherapy drugs, anticancer peptides can significantly reduce the possibility of drug resistance and effectively kill drug-resistant tumor cells. J. Am. Chem. Soc. 2024, 146, 11254-11265; J. Am. Chem. Soc. 2022, 144, 7283-7294.

[0004] Chinese invention patent application CN 107759678 A, published on March 6, 2018, discloses an anticancer active peptide and its application. It involves replacing K with R and E with Q in the Lycosin-I sequence (a small molecule polypeptide derived from tarantula venom), forming an active polypeptide toxin containing 23 amino acid residues. The modified active polypeptide toxin retains the selectivity of Lycosin-I and enhances its cytotoxic activity against solid tumor cells. Its long peptide structure necessitates a complex chemical synthesis process, low yield, difficult purification, and high cost for large-scale production.

[0005] In addition, fatty acid modification of some anticancer peptides can increase their activity and stability. Org. Biomol. Chem. [2015, 13, 7673-7680], and several fatty acid-modified peptide drugs have been marketed. However, fatty acid modification can easily increase the toxicity of anticancer peptides and reduce their selectivity. Toxins. 2021, 13, 867].

[0006] Finally, regarding combination therapy strategies for anticancer peptides, existing anticancer peptides exhibit limited anticancer activity and cannot effectively inhibit multiple tumor cell types. Furthermore, while most anticancer peptides demonstrate good anticancer activity, they may still be toxic to normal cells (such as erythrocytes), leading to side effects such as hemolysis. Therefore, their selectivity during use still needs further improvement. Summary of the Invention

[0007] The purpose of this invention is to provide a class of nitrogen-terminal fatty acid-modified anticancer peptides to solve the problems of complex structure and limited anticancer activity of existing anticancer peptides.

[0008] A second objective of this invention is to provide the application of the above-mentioned nitrogen-terminal fatty acid-modified anticancer peptides in the preparation of anticancer drugs, in order to solve the aforementioned problems.

[0009] To achieve the above objectives, the technical solution of the nitrogen-terminal fatty acid-modified anticancer peptide of the present invention is as follows: A class of N-terminal fatty acid-modified anticancer peptides, wherein the N-terminus of the anticancer peptide is modified with a straight-chain fatty acid, and the C-terminus is an amide structure, with the general structural formula C0. n -K a W b E c ; where C n Represents a fatty acid chain with n carbon atoms, where a is the number of lysine (K), b is the number of tryptophan (W), and c is the number of glutamic acid (E); n = 6-18; a + b + c = 8; a = 3 or 4, b = 3 or 4, and c = 0, 1, or 2.

[0010] This invention is pioneering. The nitrogen-terminal fatty acid-modified anticancer peptide is constructed by adding lysine, tryptophan, and glutamic acid to an 8-position short peptide, combined with a nitrogen-terminal fatty acid modification strategy and a carbon-terminal amide structure. Its structure is simple and its synthesis cost is low. In vitro antitumor and toxicity experiments show that this type of anticancer peptide exhibits significant antitumor activity against various tumor cells, including 4T1, HeLa, A549, and HCCLM3, demonstrating broad-spectrum anticancer activity.

[0011] In addition, some of the anticancer peptides of the present invention have low toxicity to normal cells and exhibit selectivity for tumor cells, showing good application prospects in the preparation of clinical anticancer drugs and anti-tumor multidrug resistance.

[0012] Preferably, the amino acid sequence of the anticancer peptide is selected from one of the following sequences: a) K4W4: The amino acid sequence as shown in SEQ ID NO: 1; b) K3W4E: The amino acid sequence as shown in SEQ ID NO: 2; c) K4W3E: The amino acid sequence as shown in SEQ ID NO: 3; d) K3W3E2: The amino acid sequence as shown in SEQ ID NO: 4.

[0013] Preferably, the structure of the anticancer peptide is C n -K3W3E2, n=14-18; its amino acid sequence is shown in SEQ ID NO: 4.

[0014] More preferably, the structure of the anticancer peptide is C 16 -K3W3E2 or C 18 -K3W3E2. This anticancer peptide exhibits both significant antitumor activity and low hemolytic toxicity.

[0015] Preferably, the structure of the anticancer peptide is C n -K3W4E or C n -K4W3E, where n=14-18; C n The amino acid sequence of -K3W4E is shown in SEQ ID NO: 2, C n The amino acid sequence of -K4W3E is shown in SEQ ID NO: 3.

[0016] More preferably, the structure of the anticancer peptide is C 16 -K3W4E、C 18 -K3W4E、C 16 -K4W3E or C 18 -K4W3E.

[0017] Preferably, the structure of the anticancer peptide is C n -K4W4, where n=6-18; its amino acid sequence is shown in SEQ ID NO: 1.

[0018] More preferably, the structure of the anticancer peptide is C6-K4W4. This anticancer peptide exhibits both significant antitumor activity and low hemolytic toxicity.

[0019] The above-mentioned nitrogen-terminal fatty acid-modified anticancer peptides are used in the preparation of anticancer drugs.

[0020] The nitrogen-terminal fatty acid-modified anticancer peptides of this invention have simple structures and novel designs, and can be obtained using conventional solid-phase synthesis methods, resulting in low manufacturing costs. Furthermore, these nitrogen-terminal fatty acid-modified anticancer peptides possess broad-spectrum anticancer activity and can be combined with existing anticancer drugs to achieve combined treatment of multiple cancer types.

[0021] Preferably, the anticancer drug is an anti-cervical cancer drug, an anti-non-small cell lung cancer drug, an anti-liver cancer drug, or an anti-breast cancer drug. Attached Figure Description

[0022] Figure 1The anticancer peptide C in this invention 18 - EE mass spectrum; Figure 2 This is the mass spectrum of the anticancer peptide K4W4 in this invention; Figure 3 This is the mass spectrum of the anticancer peptide C6-K4W4 in this invention; Figure 4 The anticancer peptide C in this invention 10 -K4W4 mass spectrum; Figure 5 The anticancer peptide C in this invention 14 -K4W4 mass spectrum; Figure 6 The anticancer peptide C in this invention 18 -K4W4 mass spectrum; Figure 7 The anticancer peptide C in this invention 18 -EW mass spectrum; Figure 8 The anticancer peptide C in this invention 18 -KE mass spectrum; Figure 9 This is an experimental diagram showing the antitumor activity of the nitrogen-terminal fatty acid-modified anticancer peptide of the present invention on 4T1 cells; Figure 10 This is an experimental diagram showing the antitumor activity of the nitrogen-terminal fatty acid-modified anticancer peptide of the present invention on HeLa cells; Figure 11 This is an experimental diagram showing the antitumor activity of the nitrogen-terminal fatty acid-modified anticancer peptide of the present invention on HCCLM3 cells; Figure 12 This is an experimental diagram showing the antitumor activity of the nitrogen-terminal fatty acid-modified anticancer peptide of the present invention on A549 cells; Figure 13 This is a hemolysis experiment diagram of the nitrogen-terminal fatty acid-modified anticancer peptide of the present invention; Figure 14 This is a PI staining experiment diagram of the nitrogen-terminal fatty acid modified anticancer peptide of the present invention. Detailed Implementation

[0023] (I) Preferred embodiments of the nitrogen-terminal fatty acid-modified anticancer peptides of the present invention The nitrogen-terminal fatty acid-modified anticancer peptide proposed in this invention is based on the KKWWKKWW-NH2 sequence, modified with fatty acid chains of different lengths. n By modifying its nitrogen terminus and replacing it with glutamic acid E, an anticancer peptide with simple structure, low synthesis cost, low toxicity and broad-spectrum anticancer activity was designed.

[0024] This anticancer peptide has a sequence length of 8 amino acids, composed of lysine and tryptophan, or lysine, tryptophan, and glutamic acid, with glutamic acid E located at position 1 or 8. Its general structural formula is: C n -K a W b E c .

[0025] Among them, C n This represents a fatty acid chain with n carbon atoms, where n = 6-18. It has a straight-chain fatty acid structure, with typical n values ​​of 6, 10, 12, 14, 16, and 18.

[0026] Specifically, sequence C n -KKWWKKWW-NH2, labeled as C n -K4W4, whose amino acid sequence is shown in SEQ ID NO: 1.

[0027] Sequence C n -EKWWKKWW-NH2, labeled as C n -K3W4E, its amino acid sequence is shown in SEQ ID NO: 2, where different n values ​​represent fatty acid chains of different lengths. n The nitrogen terminus is modified. The n value is preferably 14-18, more preferably 16-18, and most preferably n=16 or 18. For example, C is replaced with glutamic acid E. 18 The lysine K at position -K4W41 yields C 18 -EKWWKKWW-NH2, labeled as C 18 -K3W4E(C 18 -EW), its nitrogen terminus is modified with octadecanoic acid (i.e., 1-octadecanoic acid), that is, the nitrogen terminus fatty acid modification is completed by the coupling reaction between the carboxyl group and the amino group of octadecanoic acid.

[0028] Sequence C n -KKWWKKWE-NH2, marked as C n -K4W3E, its amino acid sequence is shown in SEQ ID NO: 3, where different n values ​​represent fatty acid chains of different lengths. n The nitrogen terminus is modified. The n value is preferably 14-18, more preferably 16-18, and most preferably n=16 or 18. For example, C is replaced with glutamic acid E. 18 -K4W48 tryptophan W, yielding C 18 -KKWWKKWE-NH2, marked as C 18 -K4W3E(C 18 -KE).

[0029] Sequence C n -EKWWKKWE-NH2, marked as Cn -K3W3E2, its amino acid sequence is shown in SEQ ID NO: 4, where different n values ​​represent fatty acid chains of different lengths. n The nitrogen-terminus is modified. The n value is preferably 14-18, more preferably 16-18, and most preferably n=16 or 18. Simultaneously, glutamic acid E is used to replace C. 18 The lysine K at position 41 and the tryptophan W at position 8 of K4W yield C. 18 -EKWWKKWE-NH2, marked as C 18 -K3W3E2(C 18 -EE).

[0030] All of the above-mentioned nitrogen-terminated fatty acid-modified anticancer peptides can be prepared using classic solid-phase synthesis methods.

[0031] The preferred embodiments described above are illustrated below with specific examples. In the following embodiments, unless otherwise specified, all raw materials involved are commercially available conventional products, and all processing techniques involved are conventional processes. Unless otherwise specified, "%" refers to percentages by mass.

[0032] Example 1 The N-terminal fatty acid-modified anticancer peptide in this embodiment has the general structural formula C0. 18 -EKWWKKWE-NH2, marked as C 18 -K3W3E2(C 18 -EE), whose amino acid sequence is shown in SEQ ID NO:4, with the nitrogen terminus modified with octadecanoic acid and the carbon terminus being an amide structure.

[0033] The preparation method of the above-mentioned nitrogen-terminal fatty acid-modified anticancer peptides adopts the following steps: (1) Resin activation and pretreatment Accurately weigh 0.476 g (0.2 mmol) of MBHA resin into a peptide synthesizer, swell the resin with DCM, stir for 30 min, wash the resin twice with DMF, and test for indole. If the resin is colorless, it indicates that the resin is normal and usable.

[0034] (2) C 18 Synthesis of EE-resin The resin that passed the test was washed with DMF containing 20% ​​piperidine to remove the Fmoc protecting group of the amino group. An indole test showed the resin was blue, indicating that the Fmoc group had been removed and the amino group was exposed. Residual piperidine was washed away with DMF. Three times the excess of Fmoc-Glu(OtBu)-OH (0.6 mmol), HOBT (0.6 mmol), and HBTU (0.6 mmol), and six times the excess of DIEA (1.2 mmol) were dissolved in DMF solution and added to a peptide synthesizer. The mixture was stirred for 1 hour under argon protection. Residual amino acids were washed away with DMF. An indole test showed the resin was colorless, indicating the formation of Fmoc-Glu-resin. The above steps were repeated to condense each amino acid sequentially until Fmoc-Glu-Lys-Trp-Trp-Lys-Lys-Trp-Glu-resin was obtained.

[0035] The resin was treated with DMF containing 20% ​​piperidine to remove the Fmoc groups. An indene test showed the resin turned blue, indicating the Fmoc groups had been removed. Residual piperidine was washed away with DMF. Three times the excess of CH3(CH2) was then added. 16 COOH (0.6 mmol), HOBT (0.6 mmol), and HBTU (0.6 mmol), along with a 6-fold excess of DIEA (1.2 mmol), were dissolved in DMF solution and added to a peptide synthesizer. The mixture was stirred and reacted for 1 hour under argon protection. Residual CH3(CH2) was then washed away with DMF. 16 COOH, indene test, the resin is colorless, yielding C. 18 -Glu-Lys-Trp-Trp-Lys-Lys-Trp-Glu-resin.

[0036] (3) Peptide cleavage The resin was washed alternately with dichloromethane and methanol, compressed, and then dried. A cutting agent (trifluoroacetic acid:triisopropylsilane:water = 9.5:0.25:0.25; volume ratio) was added, and the mixture was cut for 3 hours to obtain C. 18 -Glu-Lys-Trp-Trp-Lys-Lys-Trp-Glu-NH2 was extracted with ether and water to obtain an aqueous solution of crude peptide, which was then freeze-dried to obtain a freeze-dried powder of crude peptide.

[0037] (4) Peptide purification The synthesized crude peptide was purified by HPLC to obtain a pure peptide. During purification, the mobile phase consisted of water and acetonitrile containing 0.1% TFA (trifluoroacetic acid), with gradient elution. The liquid containing the target peak was collected and lyophilized to obtain the corresponding pure peptide.

[0038] The obtained pure peptide was analyzed by mass spectrometry, and its mass spectrum is shown below. Figure 1 As shown, the proof structure is C. 18The polypeptide of -EKWWKKWE-NH2 was successfully synthesized.

[0039] Based on this embodiment, modifications with tetradecanoic acid and hexadecanoic acid respectively can yield structures with the following C... 14 -EKWWKKWE-NH2、C 16 -EKWWKKWE-NH2 polypeptide.

[0040] Example 2 The N-terminal fatty acid-modified anticancer peptide in this embodiment has the general structural formula C0. 18 -KKWWKKWW-NH2, labeled as C 18 -K4W4, whose amino acid sequence is shown in SEQ ID NO: 1, has an octadecanoic acid modification at the nitrogen end and an amide structure at the carbon end.

[0041] The preparation method of the above-mentioned nitrogen-terminal fatty acid-modified anticancer peptides adopts the following steps: (1) Resin activation and pretreatment Accurately weigh 0.476 g (0.2 mmol) of MBHA resin into a peptide synthesizer, swell the resin with DCM, stir for 30 min, wash the resin twice with DMF, and test for indole. If the resin is colorless, it indicates that the resin is normal and usable.

[0042] (2) C 18 Synthesis of -K4W4-resin The resin that passed the above test was washed with DMF containing 20% ​​piperidine to remove the Fmoc protecting group of the amino group. An indole test showed the resin was blue, indicating that the Fmoc group had been removed and the amino group was exposed. Residual piperidine was washed away with DMF. Three times the excess of Fmoc-Trp(Boc)-OH (0.6 mmol), HOBT (0.6 mmol), and HBTU (0.6 mmol), and six times the excess of DIEA (1.2 mmol) were dissolved in DMF solution and added to a peptide synthesizer. The mixture was stirred for 1 hour under argon protection. Residual amino acids were washed away with DMF. An indole test showed the resin was colorless, indicating the formation of Fmoc-Trp-resin. The above steps were repeated to condense each amino acid sequentially until Fmoc-Lys-Lys-Trp-Trp-Lys-Lys-Trp-Trp-resin was obtained.

[0043] The resin was treated with DMF containing 20% ​​piperidine to remove the Fmoc groups. An indene test showed the resin turned blue, indicating the Fmoc groups had been removed. Residual piperidine was washed away with DMF. Three times the excess of CH3(CH2) was then added. 16COOH (0.6 mmol), HOBT (0.6 mmol), and HBTU (0.6 mmol), along with a 6-fold excess of DIEA (1.2 mmol), were dissolved in DMF solution and added to a peptide synthesizer. The mixture was stirred and reacted for 1 hour under argon protection. Residual CH3(CH2) was then washed away with DMF. 16 COOH, indene test, the resin is colorless, yielding C. 18 -Lys-Lys-Trp-Trp-Lys-Lys-Trp-Trp-resin.

[0044] (3) Peptide cleavage The resin was washed alternately with dichloromethane and methanol, compressed, and then dried. A cutting agent (trifluoroacetic acid:triisopropylsilane:water = 9.5:0.25:0.25; volume ratio) was added, and the mixture was cut for 3 hours to obtain C. 18 -Lys-Lys-Trp-Trp-Lys-Lys-Trp-Trp-NH2 was extracted with ether and water to obtain an aqueous solution of crude peptide, which was then freeze-dried to obtain a freeze-dried powder of crude peptide.

[0045] (4) Peptide purification The synthesized crude peptide was purified by HPLC to obtain a pure peptide. During purification, the mobile phase consisted of water and acetonitrile containing 0.1% TFA, with gradient elution. The liquid containing the target peak was collected and lyophilized to obtain the corresponding pure peptide.

[0046] Based on this embodiment, without fatty acid modification, a polypeptide with the structure KKWWKKWW-NH2(K4W4) was obtained, and its mass spectrum is shown below. Figure 2 As shown.

[0047] Modification with hexadecanoic acid yielded a polypeptide with the structure C6-KKWWKKWW-NH2 (C6-K4W4), and its mass spectrum is shown below. Figure 3 As shown.

[0048] Modification with decadecanoic acid yields a structure of C1. 10 -KKWWKKWW-NH2(C 10 The mass spectrum of the polypeptide (-K4W4) is shown below. Figure 4 As shown.

[0049] Modification with tetradecanoic acid yields a structure of C1. 14 -KKWWKKWW-NH2(C 14 The mass spectrum of the polypeptide (-K4W4) is shown below. Figure 5 As shown.

[0050] The mass spectrum of the pure peptide obtained by modification with octadecanoic acid (i.e., in this example) is shown below. Figure 6 As shown, the proof structure is C.18 -KKWWKKWW-NH2(C 18 The polypeptide (-K4W4) was successfully synthesized.

[0051] Example 3 The N-terminal fatty acid-modified anticancer peptide in this embodiment has the general structural formula C0. 18 -EKWWKKWW-NH2, labeled as C 18 -K3W4E(C 18 -EW), whose amino acid sequence is shown in SEQ ID NO: 2, with the nitrogen terminus modified with octadecanoic acid and the carbon terminus being an amide structure.

[0052] The preparation method of the above-mentioned nitrogen-terminal fatty acid-modified anticancer peptides adopts the following steps: (1) Resin activation and pretreatment Accurately weigh 0.476 g (0.2 mmol) of MBHA resin into a peptide synthesizer, swell the resin with DCM, stir for 30 min, wash the resin twice with DMF, and test for indole. If the resin is colorless, it indicates that the resin is normal and usable.

[0053] (2) C 18 Synthesis of -EW-resin The resin that passed the above test was washed with DMF containing 20% ​​piperidine to remove the Fmoc protecting group of the amino group. An indole test showed the resin was blue, indicating that the Fmoc group had been removed and the amino group was exposed. Residual piperidine was washed away with DMF. Three times the excess of Fmoc-Trp(Boc)-OH (0.6 mmol), HOBT (0.6 mmol), and HBTU (0.6 mmol), and six times the excess of DIEA (1.2 mmol) were dissolved in DMF solution and added to a peptide synthesizer. The mixture was stirred and reacted for 1 hour under argon protection. Residual amino acids were washed away with DMF. An indole test showed the resin was colorless, indicating the formation of Fmoc-Trp-resin. The above steps were repeated to condense each amino acid sequentially until Fmoc-Glu-Lys-Trp-Trp-Lys-Lys-Trp-Trp-resin was obtained.

[0054] The resin was treated with DMF containing 20% ​​piperidine to remove the Fmoc groups. An indene test showed the resin turned blue, indicating the Fmoc groups had been removed. Residual piperidine was washed away with DMF. Three times the excess of CH3(CH2) was then added. 16 COOH (0.6 mmol), HOBT (0.6 mmol), and HBTU (0.6 mmol), along with a 6-fold excess of DIEA (1.2 mmol), were dissolved in DMF solution and added to a peptide synthesizer. The mixture was stirred and reacted for 1 hour under argon protection. Residual CH3(CH2) was then washed away with DMF. 16COOH, indene test, the resin is colorless, yielding C. 18 -Glu-Lys-Trp-Trp-Lys-Lys-Trp-Trp-resin.

[0055] (3) Peptide cleavage The resin was washed alternately with dichloromethane and methanol, compressed, and then dried. A cutting agent (trifluoroacetic acid:triisopropylsilane:water = 9.5:0.25:0.25; volume ratio) was added, and the mixture was cut for 3 hours to obtain C. 18 -Glu-Lys-Trp-Trp-Lys-Lys-Trp-Trp-NH2 was extracted with ether and water to obtain an aqueous solution of crude peptide, which was then freeze-dried to obtain a freeze-dried powder of crude peptide.

[0056] (4) Peptide purification The synthesized crude peptide was purified by HPLC to obtain a pure peptide. During purification, the mobile phase consisted of water and acetonitrile containing 0.1% TFA, with gradient elution. The liquid containing the target peak was collected and lyophilized to obtain the corresponding pure peptide.

[0057] The obtained pure peptide was analyzed by mass spectrometry, and its mass spectrum is shown below. Figure 7 As shown, the proof structure is C. 18 The polypeptide of -EKWWKKWW-NH2 was successfully synthesized.

[0058] Based on this embodiment, modifications with tetradecanoic acid and hexadecanoic acid respectively can yield structures with the following C... 14 -EKWWKKWW-NH2、C 16 -EKWWKKWW-NH2 polypeptide.

[0059] Example 4 The N-terminal fatty acid-modified anticancer peptide in this embodiment has the general structural formula C0. 18 -KKWWKKWE-NH2, marked as C 18 -K4W3E(C 18 -KE), whose amino acid sequence is shown in SEQ ID NO: 3, with the nitrogen terminus modified with octadecanoic acid and the carbon terminus being an amide structure.

[0060] The preparation method of the above-mentioned nitrogen-terminal fatty acid-modified anticancer peptides adopts the following steps: (1) Resin activation and pretreatment Accurately weigh 0.476 g (0.2 mmol) of MBHA resin into a peptide synthesizer, swell the resin with DCM, stir for 30 min, wash the resin twice with DMF, and test for indole. If the resin is colorless, it indicates that the resin is normal and usable.

[0061] (2) C 18Synthesis of -KE-resin The resin that passed the test was washed with DMF containing 20% ​​piperidine to remove the Fmoc protecting group of the amino group. An indole test showed the resin was blue, indicating that the Fmoc group had been removed and the amino group was exposed. Residual piperidine was washed away with DMF. Three times the excess of Fmoc-Glu(OtBu)-OH (0.6 mmol), HOBT (0.6 mmol), and HBTU (0.6 mmol), and six times the excess of DIEA (1.2 mmol) were dissolved in DMF solution and added to a peptide synthesizer. The mixture was stirred for 1 hour under argon protection. Residual amino acids were washed away with DMF. An indole test showed the resin was colorless, indicating the formation of Fmoc-Glu-resin. The above steps were repeated to condense each amino acid sequentially until Fmoc-Lys-Lys-Trp-Trp-Lys-Lys-Trp-Glu-resin was obtained.

[0062] The resin was treated with DMF containing 20% ​​piperidine to remove the Fmoc groups. An indene test showed the resin turned blue, indicating the Fmoc groups had been removed. Residual piperidine was washed away with DMF. Three times the excess of CH3(CH2) was then added. 16 COOH (0.6 mmol), HOBT (0.6 mmol), and HBTU (0.6 mmol), along with a 6-fold excess of DIEA (1.2 mmol), were dissolved in DMF solution and added to a peptide synthesizer. The mixture was stirred and reacted for 1 hour under argon protection. Residual CH3(CH2) was then washed away with DMF. 16 COOH, indene test, the resin is colorless, yielding C. 18 -Lys-Lys-Trp-Trp-Lys-Lys-Trp-Glu-resin.

[0063] (3) Peptide cleavage The resin was washed alternately with dichloromethane and methanol, compressed, and then dried. A cutting agent (trifluoroacetic acid:triisopropylsilane:water = 9.5:0.25:0.25; volume ratio) was added, and the mixture was cut for 3 hours to obtain C. 18 -Lys-Lys-Trp-Trp-Lys-Lys-Trp-Glu-NH2 was extracted with ether and water to obtain an aqueous solution of the peptide, which was then freeze-dried to obtain a lyophilized peptide powder.

[0064] (4) Peptide purification The synthesized crude peptide was purified by HPLC to obtain a pure peptide. During purification, the mobile phase consisted of water and acetonitrile containing 0.1% TFA, with gradient elution. The liquid containing the target peak was collected and lyophilized to obtain the corresponding pure peptide.

[0065] The obtained pure peptide was analyzed by mass spectrometry, and its mass spectrum is shown below. Figure 8 As shown, the proof structure is C.18 The polypeptide of -KKWWKKWE-NH2 was successfully synthesized.

[0066] Based on this embodiment, modifications with tetradecanoic acid and hexadecanoic acid respectively can yield structures with the following C... 14 -KKWWKKWE-NH2、C 16 -KKWWKKWE-NH2 peptide.

[0067] (II) Preferred embodiments of the application of the nitrogen-terminal fatty acid-modified anticancer peptides of the present invention in the preparation of anticancer drugs. The aforementioned anticancer peptides modified with nitrogen-terminal fatty acids can kill tumor cells by disrupting the tumor cell membrane. This mechanism of action makes them promising for overcoming multidrug resistance in tumor cells.

[0068] These anticancer peptides exhibit significant antitumor activity against tumor cells 4T1, HeLa, A549, and HCCLM3. Some of these peptides also possess low hemolytic toxicity, such as C... n -K3W3E2, C6-K4W4, etc., indicate that they have good selectivity and can be used as clinical therapeutic drugs.

[0069] Based on the aforementioned anticancer peptides, antitumor drugs can be formulated by combining them with pharmaceutically acceptable carriers, diluents, or excipients in accordance with conventional methods in the field.

[0070] Antitumor drugs containing the above-mentioned anticancer peptides can be prepared in any form, such as oral dosage forms, injections, or topical formulations. These formulations can preferably be prepared for oral and injectable administration (true solutions, suspensions, or emulsions), with oral administration being the most preferred, such as tablets, capsules, soft capsules, liquid preparations, pills, granules, etc.

[0071] In the preparation of the above formulations, the anticancer peptides can be filled into soft capsules without any excipients, or the anticancer peptides can be mixed with a carrier or diluted with a carrier to form a suitable formulation. Examples of suitable carriers include starch, water, saline, Ringer's solution, and glucose.

[0072] Common drug diluents include saline, buffered saline, glucose, water, glycerol, and ethanol, or any combination thereof.

[0073] Depending on the purpose of medication and the specific disease, drugs containing the aforementioned anticancer peptides can be administered in different ways. Generally, the actual amount of active ingredient administered should be determined based on various relevant factors, including the disease to be treated, the severity of the patient's symptoms, other medications being taken concurrently (e.g., chemotherapy agents), and the patient's individual age, sex, weight, diet, timing of medication, and chosen route of administration. The dosage and route of administration of the aforementioned antitumor drugs can be adjusted according to the type and severity of the disease.

[0074] Antitumor drugs containing the aforementioned anticancer peptides can be administered orally or parenterally. Parenteral administration refers to the administration of drugs through routes other than oral administration, including rectal, intravenous, intraperitoneal, intramuscular, intra-arterial, transdermal, intranasal, inhalation, ocular, and subcutaneous routes.

[0075] The preferred embodiments are illustrated below with specific examples.

[0076] Example 5 The application of the nitrogen-terminal fatty acid-modified anticancer peptide in the preparation of anticancer drugs in this embodiment is described in detail below: 1. Cell Culture 4T1 and HeLa cells were cultured in 1640 with 10% FBS, while HCCLM3 and A549 cells were cultured in DMEM with 10% FBS. All cells were cultured in a humid environment at 37°C with 5% carbon dioxide.

[0077] 2. In vitro antitumor experiments and toxicity to normal cells—hemolysis experiments 1) In vitro antitumor experiment: The MTT assay was used to determine the toxicity of the above-mentioned anticancer peptides against various tumor cell lines (4T1, HeLa, HCCLM3, A549) and to evaluate their antitumor activity. Cells were seeded in 96-well plates at 5000 cells / well and cultured for 24 h. Then, different concentrations of the drug (prepared with the corresponding cell culture medium) were co-incubated with the cells for 24 h. 10 μL of MTT was added to each well, and incubation continued for another 4 h. The liquid in the wells was discarded, and 150 μL of DMSO was added to each well. After mixing, the absorbance at 490 nm was measured using a microplate reader, and cell viability was calculated. Wells containing only culture medium served as blank controls. Three independent parallel experiments were performed, and the results are shown below. Figures 9-12 As shown.

[0078] Cell viability (%) = mean absorbance of drug-treated wells ÷ mean absorbance of blank control wells × 100%.

[0079] 2) Toxicity to normal cells—hemolysis test: Mouse blood was collected, centrifuged at 800g for 5 min, the supernatant was discarded, and red blood cells were collected. The red blood cells were washed three times with PBS and resuspended in PBS to obtain an 8% (v / v) red blood cell suspension. Different concentrations (12.5, 25, 50, 100, 200 μM) of the drug (prepared with PBS) and red blood cells were co-incubated at 37℃ for 1 h, centrifuged at 1200g for 15 min, and 100 μL of the supernatant was transferred to a new 96-well plate. The absorbance at 490 nm was measured using a microplate reader to calculate the hemolysis rate. Wells treated with 2% Triton served as positive controls, and wells treated with PBS served as negative controls. Three independent parallel experiments were performed, and the results are shown below. Figure 13 As shown.

[0080] The hemolysis rate (%) is calculated as follows: (absorbance of experimental group - absorbance of negative control group) / (absorbance of positive control group - absorbance of negative control group) × 100%.

[0081] The IC50 of various anticancer peptides on tumor cells 50 Values ​​and the Hc values ​​of various anticancer peptides on erythrocytes 50 The values ​​of peptide concentrations that cause 50% hemolysis of red blood cells are summarized in Table 1.

[0082] Table 1 IC50 of anticancer peptides on tumor cells 50 Values ​​and Hc values ​​for red blood cells 50 value

[0083] a IC 50 The value represents the concentration of anticancer peptides that inhibit the growth of 50% of tumor cells.

[0084] b HC 50 The value is the concentration of anticancer peptides that causes hemolysis in 50% of red blood cells.

[0085] As shown in Table 1, the unmodified anticancer peptide K4W4 exhibits poor anticancer activity. After C... 6-18 After fatty acid modification, the inhibitory activity of the anticancer peptides on 4T1, HeLa, HCCLM3, and A549 cells was significantly enhanced, with C being the most abundant. 14-18 Fatty acid-modified anticancer peptides exhibit superior inhibitory activity. Furthermore, C6-K4W4 and C... 10 -K4W4、C 14 -K4W4、C 18 -K4W4、C 18 -EW, C 18 -KE、C 18 -EE anticancer peptides caused hemolysis of 50% of red blood cells at concentrations higher than their effective therapeutic concentrations for tumor cells, demonstrating selective toxicity to tumor cells.

[0086] C was obtained through an amino acid substitution strategy. 18 -EW, C 18 -KE、C 18 -EE anticancer peptides exhibit superior selective toxicity, especially C. 18 -EE, its HC 50 The values ​​were 4T1, HeLa, HCCLM3, and A549 cell IC50 values, respectively. 50 The values ​​were 16.3 times, 9.3 times, 19.8 times, and 6.2 times higher, demonstrating excellent selectivity for tumor cells. Meanwhile, C6-K4W4 also exhibited excellent selectivity in tumor cell inhibition experiments.

[0087] 3. PI staining experiment HCCLM3 cells were seeded in small glass dishes at a density of 40,000 cells / dish and cultured for 24 hours. The original culture medium was then discarded, and a solution containing 20 μM C ​​was added. 18 Cells were cultured in EE medium for 24 h, the drug-containing medium was discarded, and the cells were washed once with PBS. Cells were then stained with 50 μg / mL propidium iodide (PI) in the dark for 5 min, the staining solution was discarded, and 1 mL of PBS was added to each well. Cell staining was observed using a laser confocal microscope. Cells not treated with the drug served as a control. Results are as follows: Figure 14 As shown.

[0088] PI cannot pass through a normal cell membrane. When the cell membrane is damaged, PI can pass through the damaged cell membrane, enter the cell, bind to DNA, and exhibit red fluorescence. Figure 14 The results showed that, compared to the control group, C 18 The group with -EE action exhibits significant red fluorescence, indicating that this type of anticancer peptide can significantly disrupt the cell membrane of tumor cells, thereby killing tumor cells. This unique mechanism of action makes it have great potential in overcoming multidrug resistance in tumors.

[0089] The above experiments demonstrate that the nitrogen-terminal fatty acid-modified anticancer peptides of this invention have simple structures and novel designs, and can be obtained using conventional solid-phase synthesis methods, resulting in low manufacturing costs. Furthermore, these anticancer peptides exhibit significant antitumor activity and low hemolytic toxicity against tumor cells 4T1, HeLa, A549, and HCCLM3, showing promising applications in the preparation of clinical antitumor drugs and the treatment of multidrug resistance in tumors.

[0090] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A class of anticancer peptides modified with nitrogen-terminal fatty acids, characterized in that, The nitrogen terminus of the anticancer peptide is modified with a straight-chain fatty acid, and the carbon terminus is an amide structure with the general structural formula C0. n -K a W b E c ; where C n Represents a fatty acid chain with n carbon atoms, where a is the number of lysine (K), b is the number of tryptophan (W), and c is the number of glutamic acid (E); n = 6-18; a + b + c = 8; a = 3 or 4, b = 3 or 4, and c = 0, 1, or 2.

2. The nitrogen-terminal fatty acid-modified anticancer peptide as described in claim 1, characterized in that, The amino acid sequence of the anticancer peptide is selected from one of the following sequences: a) K4W4: The amino acid sequence as shown in SEQ ID NO: 1; b) K3W4E: The amino acid sequence as shown in SEQ ID NO: 2; c) K4W3E: The amino acid sequence as shown in SEQ ID NO: 3; d) K3W3E2: The amino acid sequence as shown in SEQ ID NO:

4.

3. The nitrogen-terminal fatty acid-modified anticancer peptide as described in claim 1, characterized in that, The structure of the anticancer peptide is C. n -K3W3E2, n=14-18; its amino acid sequence is shown in SEQ ID NO:

4.

4. The nitrogen-terminal fatty acid-modified anticancer peptide as described in claim 3, characterized in that, The structure of the anticancer peptide is C. 16 -K3W3E2 or C 18 -K3W3E2.

5. The nitrogen-terminal fatty acid-modified anticancer peptide as described in claim 1, characterized in that, The structure of the anticancer peptide is C. n -K3W4E or C n -K4W3E, where n=14-18; C n The amino acid sequence of -K3W4E is shown in SEQ ID NO: 2, C n The amino acid sequence of -K4W3E is shown in SEQ ID NO:

3.

6. The nitrogen-terminal fatty acid-modified anticancer peptide as described in claim 5, characterized in that, The structure of the anticancer peptide is C. 16 -K3W4E、C 18 -K3W4E、C 16 -K4W3E or C 18 -K4W3E.

7. The nitrogen-terminal fatty acid-modified anticancer peptide according to claim 1, characterized in that, The structure of the anticancer peptide is C. n -K4W4, where n=6-18; its amino acid sequence is shown in SEQ ID NO:

1.

8. The nitrogen-terminal fatty acid-modified anticancer peptide as described in claim 7, characterized in that, The structure of the anticancer peptide is C6-K4W4.

9. The use of an anticancer peptide modified with nitrogen-terminated fatty acid as described in any one of claims 1-8 in the preparation of anticancer drugs.

10. The application as described in claim 9, characterized in that, The anticancer drugs mentioned are anti-cervical cancer drugs, anti-non-small cell lung cancer drugs, anti-liver cancer drugs, or anti-breast cancer drugs.

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