Anti-cancer oligopeptide designed based on threonine and analogues thereof and application of anti-cancer oligopeptide

Abstract

The invention discloses an anti-cancer oligopeptide designed based on threonine and analogues thereof and application of the anti-cancer oligopeptide, the anti-cancer oligopeptide is obtained by taking KLLKKLLKKLLKKKLLKW as a structural basis, replacing amino acids at different sites with hydroxyl-containing threonine or analogues thereof, and then amidating a C terminal; the structural general formula of the compound is as follows: KXmLKKLLKKLLKW-NH2, wherein X = T, pT, F, Y or pY, and p represents phosphorylation; and m is 2, 3, 6, 7, 10, 11 or 13. The anti-cancer oligopeptide has the advantages of high efficiency, low toxicity, short sequence and the like. In-vitro anti-tumor activity and toxicity experiment results show that the compound has a relatively strong killing effect on various tumor cells, and is low in hemolytic toxicity and high in therapeutic index. Serum stability experiments further show that the protein has relatively high enzymolysis stability. A scanning electron microscope experiment shows that the anti-cancer oligopeptide can quickly kill tumor cells through an effective membrane rupture mechanism. Therefore, the compound has a good application prospect in the aspects of research on novel high-efficiency low-toxicity polypeptide anti-cancer drugs, resistance to multidrug resistance and preparation of anti-cancer 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 based on threonine and its analogues. This invention also relates to the application of these anticancer short peptides in the preparation of clinical antitumor drugs and in combating multidrug resistance in tumors. Background Technology

[0002] Cancer is a complex disease characterized by the malignant proliferation and spread of cells. Medically, it is known as malignant tumors and is one of the major public health problems worldwide, accounting for approximately one-sixth of all deaths globally [IEEE J. Biomed. Health 2025, 29(3): 1564-1566]. Currently, common clinical treatments for cancer include radiotherapy, chemotherapy, surgery, and a combination of surgery and chemotherapy [Nature Rev. Clin. Oncol. 2020, 17(2): 91-10]. Although the combination of chemotherapy drugs and surgery has shown good therapeutic effects, it lacks specificity, thus producing strong side effects on normal tissue cells and is costly [Nature Rev. Clin. Oncol. 2020, 17(2): 91-10]. Therefore, there is an urgent need for a new class of anticancer drugs with low toxicity that can effectively combat multidrug resistance in tumors.

[0003] Anticancer peptides (ACPs) are a class of bioactive peptides with broad-spectrum antitumor activity and low drug resistance, typically composed of 10-50 amino acids. Studies have shown that cationic amphiphilic anticancer peptides can bind to and perturb the membranes of anionic cancer cells through electrostatic adsorption and hydrophobic interactions, thereby exerting highly efficient antitumor activity and exhibiting low toxicity to normal tissue cells, demonstrating good selectivity [IEEE J. Biomed. Health 2025, 29(3): 1714-1725; Curr. Pharm. Biotechnol. 2025, 12:1153–1165]. Furthermore, compared to traditional anticancer drugs, the membrane-cleaving effect of anticancer peptides results in lower drug resistance and effective resistance to multidrug resistance [Mini-Rev. Med. Chem. 2015, 15: 73-81]. Nevertheless, existing anticancer peptides still suffer from a series of problems such as long sequences, high toxicity, and poor stability, limiting their clinical application and development. Summary of the Invention

[0004] One of the objectives of this invention is to provide a class of anticancer short peptides based on threonine and its analogues, which have novel structures, short sequences, low manufacturing costs, high antitumor activity, and low toxicity.

[0005] 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.

[0006] The third objective of this invention is to provide the application of the above-mentioned anticancer short peptides in combating multidrug resistance in tumors.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: I. Structural Design of Anticancer Short Peptides Based on Threonine and its Analogs The anticancer short peptide designed based on threonine and its analogues provided by this invention is based on the structure KLLKKLLKKLLKW, with amino acids at different sites replaced by threonine or analogues with similar hydrophobicity, followed by C-terminal amidation; its general structural formula is as follows: KXmLKKLLKKLLKW-NH2, labeled as mX; Where X = T, pT, F, Y or pY, where pT is phosphorylated threonine and pY is phosphorylated tyrosine; m is a substitution site, where m = 2, 3, 6, 7, 10, 11 or 13.

[0008] Specifically, the anticancer short peptide designed based on threonine and its analogues according to the present invention has the following structural formula: LysXmLeuLysLysLeuLeuLysLysLeuLeuLysTrp-NH2, labeled as mX; As a preferred embodiment of the technical solution of the present invention, the above-mentioned anticancer short peptide is: 2T, whose amino acid sequence is: KTLKKLLKKLLKW-NH2, as shown in SEQ ID No. 1; Alternatively: 2pT, its amino acid sequence is: KpTLKKLLKKLLKW-NH2; Or: 2F, whose amino acid sequence is: KFLKKLLKKLLKW-NH2, as shown in SEQ ID No. 2; Alternatively: 2Y, whose amino acid sequence is: KYLKKLLKKLLKW-NH2, as shown in SEQ ID No. 3; Alternatively: 2pY, its amino acid sequence is: KpYLKKLLKKLLKW-NH2; Or: 3T, whose amino acid sequence is: KLTKKLLKKLLKW-NH2, as shown in SEQ ID No. 4; Or: 6T, whose amino acid sequence is: KLLKKTLKKLLKW-NH2, as shown in SEQ ID No. 5; Or: 7T, whose amino acid sequence is: KLLKKLTKKLLKW-NH2, as shown in SEQ ID No. 6; Or: 10T, whose amino acid sequence is: KLLKKLLKKTLKW-NH2, as shown in SEQ ID No. 7; Or: 11T, whose amino acid sequence is: KLLKKLLKKLTKW-NH2, as shown in SEQ ID No. 8; Or: 13T, whose amino acid sequence is: KLLKKLLKKLLKT-NH2, as shown in SEQ ID No. 9.

[0009] The anticancer short peptide is more preferably 2T, 11T or 2pT with low hemolytic toxicity; the most preferred is 2pT with higher enzymatic stability.

[0010] The aforementioned anticancer short peptides designed based on threonine and its analogues were all obtained using classical solid-phase synthesis methods.

[0011] II. Applications of anticancer short peptides designed based on threonine and its analogues 1. In vitro antitumor experiment The MTT assay was used to evaluate the in vitro antitumor activity of the anticancer short peptide of the present invention. The specific steps were as follows: tumor cells HeLa, H1975, MCF-7, and RG were subjected to a concentration of 1×10⁻⁶. 4 The cells were seeded at the specified density in 96-well plates and incubated overnight at 37 °C with 5% CO2. Different concentrations of peptides (2.5 μM, 5 μM, 10 μM, 20 μM, 40 μM) were then added, and incubation continued for 1 h. Subsequently, 10 μL of MTT solution (5 mg / mL) was added to each well under dark conditions, and incubation continued for 4 h. The original liquid in the wells was discarded, and 150 μL of LDMSO was added to each well, followed by shaking for 10 min to completely dissolve the crystals. Finally, the absorbance was measured at 490 nm using a multi-sensor microplate reader, and cell viability was calculated using the following formula: Cellviability (%) = OD peptide / OD control × 100%. Wherein, OD peptide : Absorbance at 490 nm after peptide treatment of cells; OD control The absorbance at 490 nm is the result of treating cells with a peptide-free blank culture medium. All experiments were independently repeated three times, with four parallel replicates per well. The experimental results are as follows: Figure 1 As shown.

[0012] The experimental results show that the anticancer short peptide of the present invention has a significant killing effect on a variety of tumor cells, indicating that it has good anti-tumor activity.

[0013] 2. Hemolysis test The biosafety of the peptide was assessed by detecting the release of hemoglobin from mouse erythrocytes under the influence of the peptide. The specific steps were as follows: Mouse ocular arterial blood was collected in an EP tube containing sodium heparin, centrifuged at 800 g for 5 min at 4 °C, and the erythrocytes were washed with PBS until the supernatant was clear. An 8% (v / v) erythrocyte suspension was prepared using PBS. Then, 100 μL of the erythrocyte suspension and 100 μL of peptide solutions of different concentrations were added to each well of a 96-well plate. The plates were incubated at 37 °C for 1 h. After incubation, the 96-well plates were centrifuged at 1200 g for 15 min. After centrifugation, 100 μL of supernatant was aspirated from each well and transferred to a new 96-well plate. The absorbance at 490 nm was measured using a multi-mode microplate reader. The 2% Triton X-100 group and the PBS group served as the positive control (100% hemolysis) and negative control (0% hemolysis), respectively. The peptide concentration that induced 10% hemolysis was considered the minimum toxic concentration of the peptide molecule. The experiment was independently repeated three times, with four replicates each time. Results are as follows... Figure 2 As shown.

[0014] Figure 2 The results showed that the hemolytic toxicity concentrations of the anticancer short peptides of the present invention were all significantly higher than the effective concentrations for antitumor activity. Among them, the anticancer short peptides 2T, 2pT, 2pY, 3T, 6T, 7T, 10T, 11T and 13T all showed significantly low hemolytic toxicity. In particular, 2T, 11T and 2pT had hemolytic toxicity concentrations far higher than the effective concentrations for antitumor activity, showing a high therapeutic index.

[0015] 3. Scanning electron microscopy experiment We further investigated the membrane-permeable effect of the invented anticancer short peptide using scanning electron microscopy. Tumor cells H1975 were subjected to a 1.5 × 10⁻⁶ ppm... 5 Inoculate each well in a 24-well plate with a round coverslip and incubate overnight. The next day, remove the original culture medium and add 1 mL of 1×IC to each well. 50 2 × IC 50Cells were cultured in medium containing different concentrations of peptides for 1 h. Then, the cells were washed twice with PBS. Next, 1 mL of 2.5% glutaraldehyde was added, and the cells were fixed overnight at 4 °C. On the third day, the glutaraldehyde was aspirated, and the cells were washed twice with PBS for 5 min each time. The cells were then dehydrated with ethanol solutions of 50%, 70%, 80%, 90%, and 100% concentrations for 15 min each time. After dehydration, 1 mL of tert-butanol was added to each well, and the cells were incubated at room temperature for 2 h. The tert-butanol was then aspirated, the cells were air-dried at room temperature, sputter-coated with gold, and finally, the cell morphology was observed under a scanning electron microscope. The cell group receiving only blank medium served as a blank control group. Results are as follows: Figure 3 As shown.

[0016] from Figure 3 The results show that, compared to the blank control group, the anticancer short peptide of this invention can rapidly disturb the cell membrane in a short time, causing cell membrane rupture and leakage of contents, and the degree of cell membrane damage increases with increasing concentration. The highly efficient membrane-breaking mechanism exhibited by the anticancer short peptide of this invention gives it a unique advantage in combating multidrug resistance in clinical practice.

[0017] 4. Serum stability test Mouse serum was used, and the peptide was prepared into a 10 mM solution with PBS. This solution was then mixed with the serum at a 1:2 ratio and incubated in a 37°C water bath. Samples were taken at 0 h, 0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h, 10 h, 12 h, and 24 h in ice-cold EP tubes. The reaction was immediately terminated by adding an equal volume of ice-cold acetonitrile to the peptide solution, followed by an ice bath for 15 min. The tubes were centrifuged at 13000 g at 4°C until the supernatant was clear, and the samples were collected. The collected samples were analyzed by RP-HPLC (Waters, MA, USA) to determine the remaining amount. The mobile phase consisted of 0.01% acetonitrile and deionized water, with a gradient of 5%–95% acetonitrile, for 30 min at a flow rate of 1.0 mL / min. The experiment was independently repeated three times. The results are as follows: Figure 4 As shown.

[0018] Figure 4 The results showed that, compared with threonine, the threonine hydroxyphosphorylated analog of the present invention has better resistance to protease degradation and exhibits higher serum stability.

[0019] The advantages of this invention compared to the prior art are as follows: 1. The anticancer short peptide of this invention has a novel structure, short sequence, low molecular weight, and low manufacturing cost, and can be obtained by conventional solid-phase synthesis methods.

[0020] 2. The positive charge and hydrophobicity of anticancer peptides are two major structural factors affecting their antitumor activity. When cationic anticancer peptides adsorb and bind to anionic tumor cell membranes through electrostatic interactions, moderate hydrophobicity is a key factor in promoting the selective destruction of tumor cell membranes while maintaining low toxicity. Compared with leucine, threonine, containing a hydroxyl group, has moderate hydrophobicity and can promote hydrogen bonding between anticancer peptides and tumor cell membranes, showing greater potential for improving the selectivity of anticancer peptides. In addition, different sites in the amphiphilic hydrophobic region of anticancer peptides have different abilities to penetrate and perturb lipid membranes. Further optimization of modification sites while maintaining the amphiphilic structure is more beneficial for obtaining short anticancer peptides with high activity and low toxicity. Therefore, this invention uses the leucine-rich and highly toxic anticancer short peptide KLLKKLLKKLLKW as its structural basis. By replacing different sites with threonine containing hydroxyl groups or analogs with similar hydrophobicity, the resulting anticancer short peptide preferably exhibits significant killing effects on various tumor cells (HeLa, MCF-7, RG, and H1975) and shows low hemolytic toxicity. In particular, the 2pT peptide has higher enzymatic stability and has great application potential in the preparation of clinical antitumor drugs and in combating multidrug resistance. Attached Figure Description

[0021] Figure 1 This is a diagram illustrating the in vitro antitumor activity of the anticancer short peptide of this invention against HeLa, H1975, MCF-7, and RG cells. Figure 2 This is a diagram of the hemolysis experiment of the anticancer short peptide of this invention; Figure 3 This is a scanning electron microscope image of the anticancer short peptide of this invention; Figure 4 This is a serum stability experiment diagram of the anticancer short peptide of the present invention; Figure 5 This is the mass spectrum of the anticancer short peptide 2T of the present invention; Figure 6 This is the mass spectrum of the anticancer short peptide 2pT of this invention; Figure 7 This is the mass spectrum of the anticancer short peptide 2F of this invention; Figure 8 This is the mass spectrum of the anticancer short peptide 2Y of the present invention; Figure 9 This is the mass spectrum of the anticancer short peptide 2pY of this invention; Figure 10 This is the mass spectrum of the anticancer short peptide 3T of this invention; Figure 11 This is the mass spectrum of the anticancer short peptide 6T of this invention; Figure 12 This is the mass spectrum of the anticancer short peptide 7T of this invention; Figure 13This is the mass spectrum of the anticancer short peptide 10T of this invention; Figure 14 This is the mass spectrum of the anticancer short peptide 11T of this invention; Figure 15 This is the mass spectrum of the anticancer short peptide 13T of this invention. Detailed Implementation

[0022] The synthesis process of the anticancer short peptide of the present invention will be further explained below through specific embodiments.

[0023] Example 1: Synthesis of 2T 1) Resin pretreatment Calculate and weigh the appropriate amount of resin (0.25 mmol, 0.521 g), add the resin to the bottom of the synthesizer, add dichloromethane (DCM), and stir for 30 min to allow the resin to fully swell. Wash the resin three times with an appropriate amount of DMF (N,N-dimethylformamide). Then, use the ninhydrin colorimetric method (8% phenol:pyridine:6% ninhydrin = 1:2:1). After heating for 1 min, observe the resin; if the resin is colorless, it indicates that the resin is normal and the next step can be performed.

[0024] 2) Deprotection Add the deprotecting agent (piperidine:DBU:DMF = 1:1:18), stir four times for 5 minutes each time to remove the Fmoc protecting group. A color change (blue or orange) in the resin, detected by the ninhydrin method, indicates that the Fmoc protecting group has been completely removed. Continue washing the resin with DMF to ensure the deprotecting agent is completely removed before proceeding to the next step of amino acid condensation.

[0025] 3) Amino acid condensation reaction Based on the amount of resin and the molecular weight of the amino acids, weigh out 3 times the excess of Fmoc-protected tryptophan, HOBT, HBTU and 6 times the excess of DIEA, dissolve them in DMF and add them to the synthesizer. Stir for 1 h, wash three times with DMF and detect the resin using the ninhydrin detection method. If the resin is colorless, it indicates that Fmoc-Trp-resin has been successfully synthesized.

[0026] Repeat steps 1), 2), and 3) above to continue the synthesis of the next amino acid until Lys(Boc)-Thr(Boc)-Leu-Lys(Boc)-Lys(Boc)-Leu-Leu-Lys(Boc)-Lys(Boc)-Leu-Leu-Lys(Boc)-Trp(Boc)-resin is synthesized. Then, use a deprotecting agent to remove the Fmoc group at the end of the peptide chain to obtain 2T-resin.

[0027] 4) Peptide cleavage The resin obtained above was washed with DCM and MeOH respectively. After washing, the liquid was dried until the resin became powder. A cleaving agent (TFA: Triisopropylsilane: H2O = 9.5: 0.25: 0.25 v:v:v) was added and stirred for 4 h to ensure that the peptide chain was cleaved from the resin. The peptide was extracted with ether and deionized water and then freeze-dried to obtain crude peptide lyophilized powder.

[0028] 5) Purification of peptides The crude peptide lyophilized powder obtained above was separated and purified by RP-HPLC. The eluent was collected and then freeze-dried again. Mass spectrometry identification showed that the peptide was 2T with a molecular weight of 1638.14 Da. The mass spectrum is shown below. Figure 5 The amino acid sequence is: Lys-Thr-Leu-Lys-Lys-Leu-Leu-Lys-Lys-Leu-Leu-Lys-Trp-NH2.

[0029] Example 2: Synthesis of 2pT 1) Resin pretreatment, 2) Deprotection: Same as in Example 1.

[0030] 3) Amino acid condensation reaction Based on the amount of resin and the molecular weight of the amino acids, weigh out 3 times the excess of Fmoc-protected threonine, HOBT, HBTU and 6 times the excess of DIEA, dissolve them in DMF and add them to the synthesizer. Stir for 1 h, wash three times with DMF and detect the resin using the ninhydrin detection method. If the resin is colorless, it indicates that Fmoc-Thr-resin has been successfully synthesized.

[0031] Repeat steps 1), 2), and 3) above to continue the synthesis of the next amino acid until Lys(Boc)-pThr(Boc)-Leu-Lys(Boc)-Lys(Boc)-Leu-Leu-Lys(Boc)-Lys(Boc)-Leu-Leu-Lys(Boc)-Trp(Boc)-resin is synthesized. Then, use a deprotecting agent to remove the Fmoc group at the end of the peptide chain to obtain 2pT-resin.

[0032] 4) Peptide cleavage: Same as in Example 1.

[0033] 5) Purification of peptides Same as in Example 1, mass spectrometry analysis revealed a molecular weight of 1718.11 Da, with a mass spectrum shown below. Figure 6 The amino acid sequence is: Lys-pThr-Leu-Lys-Lys-Leu-Leu-Lys-Lys-Leu-Leu-Lys-Trp-NH2.

[0034] Example 3: Synthesis of 2F 1) Resin pretreatment, 2) Deprotection: Same as in Example 1.

[0035] 3) Amino acid condensation reaction Based on the amount of resin and the molecular weight of the amino acids, weigh out 3 times the excess of Fmoc-protected threonine, HOBT, HBTU and 6 times the excess of DIEA, dissolve them in DMF and add them to the synthesizer. Stir for 1 h, wash three times with DMF and detect the resin using the ninhydrin detection method. If the resin is colorless, it indicates that Fmoc-Thr-resin has been successfully synthesized.

[0036] Repeat steps 1), 2), and 3) above to continue the synthesis of the next amino acid until Lys(Boc)-Phe(Boc)-Leu-Lys(Boc)-Lys(Boc)-Leu-Leu-Lys(Boc)-Lys(Boc)-Leu-Leu-Lys(Boc)-Trp(Boc)-resin is synthesized. Then, use a deprotecting agent to remove the Fmoc group at the end of the peptide chain to obtain 2F-resin.

[0037] 4) Peptide cleavage: Same as in Example 1.

[0038] 5) Purification of peptides Same as in Example 1, 2F was identified by mass spectrometry with a molecular weight of 1684.16 Da. The mass spectrum is shown below. Figure 7 The amino acid sequence is: Lys-Phe-Leu-Lys-Lys-Leu-Leu-Lys-Lys-Leu-Leu-Lys-Trp-NH2.

[0039] Example 4: Synthesis of 2Y 1) Resin pretreatment, 2) Deprotection: Same as in Example 1.

[0040] 3) Amino acid condensation reaction Based on the amount of resin and the molecular weight of the amino acids, weigh out 3 times the excess of Fmoc-protected threonine, HOBT, HBTU and 6 times the excess of DIEA, dissolve them in DMF and add them to the synthesizer. Stir for 1 h, wash three times with DMF and detect the resin using the ninhydrin detection method. If the resin is colorless, it indicates that Fmoc-Thr-resin has been successfully synthesized.

[0041] Repeat steps 1), 2), and 3) above to continue the synthesis of the next amino acid until Lys(Boc)-Tyr(Boc)-Leu-Lys(Boc)-Lys(Boc)-Leu-Leu-Lys(Boc)-Lys(Boc)-Leu-Leu-Lys(Boc)-Trp(Boc)-resin is synthesized. Then, use a deprotecting agent to remove the Fmoc group at the end of the peptide chain to obtain 2Y-resin.

[0042] 4) Peptide cleavage: Same as in Example 1.

[0043] 5) Purification of peptides Same as in Example 1, 2Y was identified by mass spectrometry as having a molecular weight of 1700.16 Da. The mass spectrum is shown below. Figure 8 The amino acid sequence is: Lys-Tyr-Leu-Lys-Lys-Leu-Leu-Lys-Lys-Leu-Leu-Lys-Trp-NH2.

[0044] Example 5: Synthesis of 2pY 1) Resin pretreatment, 2) Deprotection: Same as in Example 1.

[0045] 3) Amino acid condensation reaction Based on the amount of resin and the molecular weight of the amino acids, weigh out 3 times the excess of Fmoc-protected threonine, HOBT, HBTU and 6 times the excess of DIEA, dissolve them in DMF and add them to the synthesizer. Stir for 1 h, wash three times with DMF and detect the resin using the ninhydrin detection method. If the resin is colorless, it indicates that Fmoc-Thr-resin has been successfully synthesized.

[0046] Repeat steps 1), 2), and 3) above to continue the synthesis of the next amino acid until Lys(Boc)-pTyr(Boc)-Leu-Lys(Boc)-Lys(Boc)-Leu-Leu-Lys(Boc)-Lys(Boc)-Leu-Leu-Lys(Boc)-Trp(Boc)-resin is synthesized. Then, use a deprotecting agent to remove the Fmoc group at the end of the peptide chain to obtain 2pY-resin.

[0047] 4) Peptide cleavage: Same as in Example 1.

[0048] 5) Purification of peptides Same as in Example 1, the 2pY was identified by mass spectrometry with a molecular weight of 1781.13 Da. The mass spectrum is shown below. Figure 9 The amino acid sequence is: Lys-pTyr-Leu-Lys-Lys-Leu-Leu-Lys-Lys-Leu-Leu-Lys-Trp-NH2.

[0049] Example 6: Synthesis of 3T 1) Resin pretreatment, 2) Deprotection: Same as in Example 1.

[0050] 3) Amino acid condensation reaction Based on the amount of resin and the molecular weight of the amino acids, weigh out 3 times the excess of Fmoc-protected tryptophan, HOBT, HBTU and 6 times the excess of DIEA, dissolve them in DMF and add them to the synthesizer. Stir for 1 h, wash three times with DMF and detect the resin using the ninhydrin detection method. If the resin is colorless, it indicates that Fmoc-Trp-resin has been successfully synthesized.

[0051] Repeat steps 1), 2), and 3) above to continue the synthesis of the next amino acid until the synthesis is complete. Lys(Boc)-Leu-Thr(Boc)-Lys(Boc)-Lys(Boc)-Leu-Leu-Lys(Boc)-Lys(Boc)-Leu-Leu-Lys(Boc)-Trp(Boc)-resin, then the Fmoc group at the end of the peptide chain is removed with a deprotecting agent to obtain 3T-resin.

[0052] 4) Peptide cleavage: Same as in Example 1.

[0053] 5) Purification of peptides Same as in Example 1, the 3T was identified by mass spectrometry as having a molecular weight of 1638.14 Da. The mass spectrum is shown below. Figure 10 The amino acid sequence is: Lys-Leu-Thr-Lys-Lys-Leu-Leu-Lys-Lys-Leu-Leu-Lys-Trp-NH2.

[0054] Example 7: Synthesis of 6T 1) Resin pretreatment, 2) Deprotection: Same as in Example 1.

[0055] 3) Amino acid condensation reaction Based on the amount of resin and the molecular weight of the amino acids, weigh out 3 times the excess of Fmoc-protected tryptophan, HOBT, HBTU and 6 times the excess of DIEA, dissolve them in DMF and add them to the synthesizer. Stir for 1 h, wash three times with DMF and detect the resin using the ninhydrin detection method. If the resin is colorless, it indicates that Fmoc-Trp-resin has been successfully synthesized.

[0056] Repeat steps 1), 2), and 3) above to continue the synthesis of the next amino acid until the synthesis is complete. Lys(Boc)-Leu-Leu-Lys(Boc)-Lys(Boc)-Thr(Boc)-Leu-Lys(Boc)-Lys(Boc)-Leu-Leu-Lys(Boc)-Trp(Boc)-resin, then the Fmoc group at the end of the peptide chain is removed with a deprotecting agent to obtain 6T-resin.

[0057] 4) Peptide cleavage: Same as in Example 1.

[0058] 5) Purification of peptides Same as in Example 1, the 6T was identified by mass spectrometry as having a molecular weight of 1638.14 Da. The mass spectrum is shown below. Figure 11 The amino acid sequence is: Lys-Leu-Leu-Lys-Lys-Thr-Leu-Lys-Lys-Leu-Leu-Lys-Trp-NH2.

[0059] Example 8: Synthesis of 7T 1) Resin pretreatment, 2) Deprotection: Same as in Example 1.

[0060] 3) Amino acid condensation reaction Based on the amount of resin and the molecular weight of the amino acids, weigh out 3 times the excess of Fmoc-protected tryptophan, HOBT, HBTU and 6 times the excess of DIEA, dissolve them in DMF and add them to the synthesizer. Stir for 1 h, wash three times with DMF and detect the resin using the ninhydrin detection method. If the resin is colorless, it indicates that Fmoc-Trp-resin has been successfully synthesized.

[0061] Repeat steps 1), 2), and 3) above to continue the synthesis of the next amino acid until the synthesis is complete. Lys(Boc)-Leu-Leu-Lys(Boc)-Lys(Boc)-Leu-Thr(Boc)-Lys(Boc)-Lys(Boc)-Leu-Leu-Lys(Boc)-Trp(Boc)-resin, then the Fmoc group at the end of the peptide chain is removed with a deprotecting agent to obtain 7T-resin.

[0062] 4) Peptide cleavage: Same as in Example 1.

[0063] 5) Purification of peptides Same as in Example 1, the 7T was identified by mass spectrometry as having a molecular weight of 1638.14 Da. The mass spectrum is shown below. Figure 12 The amino acid sequence is: Lys-Leu-Leu-Lys-Lys-Leu-Thr-Lys-Lys-Leu-Leu-Lys-Trp-NH2.

[0064] Example 9: Synthesis of 10T 1) Resin pretreatment, 2) Deprotection: Same as in Example 1.

[0065] 3) Amino acid condensation reaction Based on the amount of resin and the molecular weight of the amino acids, weigh out 3 times the excess of Fmoc-protected tryptophan, HOBT, HBTU and 6 times the excess of DIEA, dissolve them in DMF and add them to the synthesizer. Stir for 1 h, wash three times with DMF and detect the resin using the ninhydrin detection method. If the resin is colorless, it indicates that Fmoc-Trp-resin has been successfully synthesized.

[0066] Repeat steps 1), 2), and 3) above to continue the synthesis of the next amino acid until the synthesis is complete. Lys(Boc)-Leu-Leu-Lys(Boc)-Lys(Boc)-Leu-Leu-Lys(Boc)-Lys(Boc)-Thr(Boc)-Leu-Lys(Boc)-Trp(Boc)-resin, then the Fmoc group at the end of the peptide chain is removed with a deprotecting agent to obtain 10T-resin.

[0067] 4) Peptide cleavage: Same as in Example 1.

[0068] 5) Purification of peptides Same as in Example 1, mass spectrometry analysis revealed 10T with a molecular weight of 1638.14 Da. (Mass spectrum shown below.) Figure 13 The amino acid sequence is: Lys-Leu-Leu-Lys-Lys-Leu-Leu-Lys-Lys-Thr-Leu-Lys-Trp-NH2.

[0069] Example 10: Synthesis of 11T 1) Resin pretreatment, 2) Deprotection: Same as in Example 1.

[0070] 3) Amino acid condensation reaction Based on the amount of resin and the molecular weight of the amino acids, weigh out 3 times the excess of Fmoc-protected tryptophan, HOBT, HBTU and 6 times the excess of DIEA, dissolve them in DMF and add them to the synthesizer. Stir for 1 h, wash three times with DMF and detect the resin using the ninhydrin detection method. If the resin is colorless, it indicates that Fmoc-Trp-resin has been successfully synthesized.

[0071] Repeat steps 1), 2), and 3) above to continue the synthesis of the next amino acid until the synthesis is complete. Lys(Boc)-Leu-Leu-Lys(Boc)-Lys(Boc)-Leu-Leu-Lys(Boc)-Lys(Boc)-Leu-Thr(Boc)-Lys(Boc)-Trp(Boc)-resin, then the Fmoc group at the end of the peptide chain is removed with a deprotecting agent to obtain 11T-resin.

[0072] 4) Peptide cleavage: Same as in Example 1.

[0073] 5) Purification of peptides Same as in Example 1, mass spectrometry analysis revealed 11T with a molecular weight of 1638.14 Da. The mass spectrum is shown below. Figure 14 The amino acid sequence is: Lys-Leu-Leu-Lys-Lys-Leu-Leu-Lys-Lys-Leu-Thr-Lys-Trp-NH2.

[0074] Example 11: Synthesis of 13T 1) Resin pretreatment, 2) Deprotection: Same as in Example 1.

[0075] 3) Amino acid condensation reaction Based on the amount of resin and the molecular weight of the amino acids, weigh out 3 times the excess of Fmoc-protected threonine, HOBT, HBTU and 6 times the excess of DIEA, dissolve them in DMF and add them to the synthesizer. Stir for 1 h, wash three times with DMF and detect the resin using the ninhydrin detection method. If the resin is colorless, it indicates that Fmoc-Thr-resin has been successfully synthesized.

[0076] Repeat steps 1), 2), and 3) above to continue the synthesis of the next amino acid until the synthesis is complete. Lys(Boc)-Leu-Leu-Lys(Boc)-Lys(Boc)-Leu-Leu-Lys(Boc)-Lys(Boc)-Leu-Leu-Lys(Boc)-Thr(Boc)-resin, then the Fmoc group at the end of the peptide chain is removed with a deprotecting agent to obtain 13T-resin.

[0077] 4) Peptide cleavage: Same as in Example 1.

[0078] 5) Purification of peptides Same as in Example 1, the 13T was identified by mass spectrometry with a molecular weight of 1566.13 Da. The mass spectrum is shown below. Figure 15 The amino acid sequence is: Lys-Leu-Leu-Lys-Lys-Leu-Leu-Lys-Lys-Leu-Leu-Lys-Thr-NH2.

Claims

1. An anticancer short peptide designed based on threonine and its analogues, characterized in that, The anticancer short peptide is based on the structure KLLKKLLKKLLKW, with amino acids at different sites replaced by threonines containing hydroxyl groups or analogs with similar hydrophobicity, followed by C-terminal amidation; its general structural formula is as follows: KXmLKKLLKKLLKW-NH2, labeled as mX; Where X = T, pT, F, Y or pY, where pT is phosphorylated threonine and pY is phosphorylated tyrosine; m is a substitution site, where m = 2, 3, 6, 7, 10, 11 or 13.

2. The anticancer short peptide designed based on threonine and its analogues as described in claim 1, characterized in that, The anticancer short peptide is: 2T, whose amino acid sequence is shown in SEQ ID No. 1; Or: 2pT, whose amino acid sequence is: KpTLKKLLKKLLKW-NH2; Or: 2F, whose amino acid sequence is shown in SEQ ID No. 2; Or: 2Y, whose amino acid sequence is shown in SEQ ID No. 3; Or: 2pY, whose amino acid sequence is: KpYLKKLLKKLLKW-NH2; Or: 3T, whose amino acid sequence is shown in SEQ ID No. 4; Or: 6T, whose amino acid sequence is shown in SEQ ID No. 5; Or: 7T, whose amino acid sequence is shown in SEQ ID No. 6; Or: 10T, whose amino acid sequence is shown in SEQ ID No. 7; Or: 11T, whose amino acid sequence is shown in SEQ ID No. 8; Or: 13T, whose amino acid sequence is shown in SEQ ID No.

9.

3. The anticancer short peptide designed based on threonine and its analogues as described in claim 2, characterized in that, The anticancer short peptide is 2T, 11T or 2pT.

4. The anticancer short peptide designed based on threonine and its analogues as described in claim 3, characterized in that, The anticancer short peptide is 2pT.

5. The use of the anticancer short peptides designed based on threonine and its analogues as described in any one of claims 1-4 in the preparation of clinical anticancer drugs.

6. The use of the anticancer short peptides designed based on threonine and its analogues as described in any one of claims 1-4 in the treatment of multidrug resistance in tumors.

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