CN117343142A - Leucine and lysine-rich high-efficiency low-toxicity anti-tumor peptide and application thereof - Google Patents

Leucine and lysine-rich high-efficiency low-toxicity anti-tumor peptide and application thereof Download PDF

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CN117343142A
CN117343142A CN202310945080.7A CN202310945080A CN117343142A CN 117343142 A CN117343142 A CN 117343142A CN 202310945080 A CN202310945080 A CN 202310945080A CN 117343142 A CN117343142 A CN 117343142A
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leu
lys
antitumor
peptide
tumor
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倪京满
王锐
张云
吴小燕
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Lanzhou University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

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Abstract

The invention discloses a high-efficiency low-toxicity antitumor peptide rich in leucine and lysine and application thereof, belonging to the technical field of biological medicines. The antitumor peptide structure is rich in leucine and lysine, and is obtained by introducing amino acids with different properties into the carbon terminal of the antitumor peptide structure, and then carrying out C-terminal amidation. The structural general formula is as follows: LLKKBLLKKLKLX-NH 2 Where x= L, D, E, aad, asd, asu. The antitumor peptide has simple structure and low manufacturing cost. The in vitro anti-tumor experimental result shows that the anti-tumor peptide has stronger anti-tumor activity on various tumor cells. In addition, the hemolytic experiment result shows that the antitumor peptide has the characteristics of low hemolytic toxicity and high safety. Scanning electron microscope experiments show that the antitumor peptide can rapidly kill tumor cells through a membrane rupture mechanism, so that the antitumor peptide can be developedThe novel high-efficiency low-toxicity antitumor drug, combined drug and anti-multidrug resistance have great application potential in the aspect of preparing clinical application drugs and have good application prospects.

Description

Leucine and lysine-rich high-efficiency low-toxicity anti-tumor peptide and application thereof
Technical Field
The invention belongs to the technical field of biological medicines, relates to a novel high-efficiency low-toxicity antitumor peptide, and also comprises application of the novel high-efficiency low-toxicity antitumor peptide in preparing clinical antitumor drugs and in combined medication and multi-drug resistance.
Background
Malignant tumors have become the greatest threat to human health due to their high morbidity and mortality [ int.j. Cancer.2019,144 (8): 1941-1953; calif. cancer J.Clin.2021,71 (3): 209-249]. According to World Health Organization (WHO) statistics in 2019, malignancy is the first or second major cause of death in most countries population at present, global malignancy burden will continue to rise, and it is estimated that the incidence of global malignancy in 2070 will double than in 2020 [ Nat Rev Clin oncol.2021,18 (10): 663-672].
Currently, traditional cancer treatment strategies include mainly chemotherapy, radiation therapy, surgery and immunotherapy [ biomacromolecules.2021, 11 (8): 1120; J.Clin.Oncol.2020,38,496-520]. According to the information of tumor related data, researchers are developing targeted antitumor drugs, and the drugs have great progress in reducing side effects and improving drug selectivity [ nerve Regen Res.2017,12 (2): 197-200], but clinical application of chemotherapeutic drugs is severely limited because tumor cells are easy to generate drug resistance to the existing chemotherapeutic antitumor drugs [ Nat. Rev. Cancer.2013,13:714-726 ]. Thus, there is an urgent need to develop antitumor drugs that have low toxicity to normal host cells and are effective against multiple drug resistance and alleviate drug resistance.
Antitumor peptides (ACPs) are short peptides with antitumor activity, and are attracting attention because of their advantages of simple structure, easy synthesis and modification, less side effects, difficulty in causing multiple drug resistance, and the like. In recent years, rational design of novel peptides with antitumor activity based on cationic antibacterial peptides (AMPs) is a new strategy for development of antitumor drugs [ arch.pharm.res.2013,36 (11): 1302-1310)]. ACPs inhibit the growth of tumor cells and destroy tumor cell membranes, which bind to the surface of tumor cells primarily by electrostatic interactions, and hydrophobic fragments then interact with phospholipid bilayer, thereby destroying cell membranes [ FEMS Microbiol. Lett.2002,206 (2): 143-149)]. This unique membrane damage mechanism makes ACPs less likely to induce tumor cells to develop multi-drug resistance. Although the existing antitumor peptide has better clinical value in malignant tumor treatment,however, the problems of long sequence, higher toxicity and poor targeting exist, and the patentability is seriously influenced. For example, the melittin Melittin (GIGAVLKVLTTGLPALISWI KRKRQQ) has higher anti-tumor activity, can effectively overcome tumor drug resistance, and is a potential anti-tumor polypeptide candidate drug. However, its severe hemolysis and non-specific damage to normal cells severely limit its in vivo use [ Biochim Biophys Acta biomembrane.2008, 1778 (2): 357-375]The method comprises the steps of carrying out a first treatment on the surface of the The subsequent polypeptide analogue and its nanometer preparation designed by using Melittin as template have obviously reduced hemolytic activity and raised therapeutic index, but still have the disadvantages of longer sequence and complicated preparation process [ Biotechnol adv.2021,50:107769-107791 ]]. Studies report that the antitumor peptide (ACP) LL-37 (LLGDFFRKSKEKIGKEFKRIVQRIKDF LRNLVPRTES) shows better selectivity in tumor treatment, and has passed the phase 1 clinical test (NCT 02225366) at present, but the synthesis of 37 amino acids thereof obviously improves the manufacturing cost [ front. Pharmacol.2022,13]The method comprises the steps of carrying out a first treatment on the surface of the In addition, the group of Chinese academy of sciences was designed to obtain a short sequence antitumor peptide KL1 (KLLKLLKKLLKLLK-NH) 2 ) Has better anti-tumor activity (IC50=4.6 mu M, hela) but higher hemolytic toxicity (Hc10=0.52 mu M) [ J.Med. Chem.2016,59 (11): 5238-5247); J.Med.chem.2020,63 (9): 5012-5012]。
Disclosure of Invention
One of the objects of the present invention is: provides an antitumor short peptide with simple structure, low manufacturing cost, strong antitumor activity and low hemolytic toxicity.
The second object of the present invention is: provides the application of the anti-tumor short peptide in preparing clinical anti-tumor drugs.
The third object of the present invention is: provides the application of the antitumor short peptide in combination medication and anti-multidrug resistance.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
1. structural design of high-efficiency low-toxicity antitumor peptide rich in leucine and lysine
The invention provides a high-efficiency low-toxicity antitumor peptide rich in leucine and lysine, which has the following structural general formula: LLKKBLLKKKLLXLX-NH 2 Marked 14X;
wherein x= L, D, E, aad, asd, asu, aad is 2-aminoadipic acid, asd is aminopimelic acid, asu is aminosuberic acid.
Specifically, the antitumor peptide is as follows:
14L, the amino acid sequence of which is shown as SEQ ID No. 1;
or: 14D, the amino acid sequence of which is shown as SEQ ID No. 2;
or: 14E, the amino acid sequence of which is shown in SEQ ID No. 3;
or: 14Aad, the amino acid sequence of which is Leu-Leu-Lys-Lys-Leu-Leu-Lys-Lys-Leu-Leu-Lys-Lys-Leu-Aad-NH 2 ；
Or: 14Asd, the amino acid sequence of which is Leu-Leu-Lys-Lys-Leu-Leu-Lys-Lys-Leu-Leu-Lys-Lys-Leu-Asd-NH 2 ；
Or: 14Asu, the amino acid sequence of which is Leu-Leu-Lys-Lys-Leu-Leu-Lys-Lys-Leu-Leu-Lys-Lys-Leu-Asu-NH 2 。
The high-efficiency low-toxicity antitumor peptide rich in leucine and lysine is prepared by adopting a classical solid-phase synthesis method.
2. Application of high-efficiency low-toxicity antitumor peptide rich in leucine and lysine in preparation of antitumor drugs
1. In vitro anti-tumor experiment
The in vitro antitumor activity of the antitumor peptides of the present invention was evaluated by MTT assay. Tumor cells selected include MCF-7, hela, hepaRG and A549 cells. The specific method comprises the following steps: tumor cells were seeded in 96-well plates, 1×10 4 Well at 37℃and 5% CO 2 Incubate overnight in incubator. Cells were allowed to stand for 1h with serum-free medium containing different concentrations of polypeptide (2.5. Mu.M, 5. Mu.M, 10. Mu.M, 20. Mu.M, 40. Mu.M). Then 10. Mu.L MTT (5 mg/mL) was added per well under dark conditions and incubated for 4h. The medium was gently aspirated, 150 μl DMSO was added to each well and shaken for 10min in a micro-shaker. The absorbance at 490nm was measured using a multifunctional microplate reader and the cell viability was calculated. Cells of the control group were treated with a blank medium without peptide. Experiments were independently repeated three times, four at a time in parallel. The results are shown in FIG. 1。
The results in FIG. 1 show that all the antitumor peptides of the present invention have significant antitumor activity against a variety of tumor cells.
2. Hemolysis experiment
Fresh blood from healthy mice was taken, centrifuged at 800g at 4℃for 5min, the supernatant serum was discarded, and after three washes of erythrocytes with PBS, it was formulated as a suspension containing 8% erythrocytes, 100. Mu.L per well was added to a 96-well plate. Dissolving the anticancer peptide analogue in PBS, preparing peptide solutions (12.5-200 μm) with different concentrations by double dilution method, adding into 96 wells containing red blood cells in equal volume, adding cell plate into micro-oscillator, shaking for 1min, mixing, and incubating at 37deg.C for 1 hr. The cell plates were then centrifuged at 1200g for 15min, 100. Mu.L of supernatant was aspirated from each well into a new 96-well plate, and absorbance at 490nm was measured using an ELISA reader. The haemolysis rate of the peptide was calculated. The cell group with 2% Triton X-100 function served as positive control, and the cell group with PBS function served as negative control. Experiments were independently repeated three times, four at a time in parallel. The results are shown in FIG. 2.
The results in FIG. 2 show that the concentrations of the anti-tumor peptides of the invention are higher than the effective concentrations of the anti-tumor activity, wherein the anti-tumor peptides 14D, 14E, 14Aad, 14Asd and 14Asu all show lower hemolytic toxicity, and particularly the analogues 14E and 14Aad have the concentrations of the hemolytic toxicity far higher than the effective concentrations of the anti-tumor activity and show higher therapeutic indexes.
3. Scanning electron microscope experiment
Tumor cells MCF-7 were assayed at 2X 10 per well 5 Individual cells were seeded in 24 well plates with circular coverslips and incubated at 37℃and 5% CO 2 Incubate overnight in incubator. The original medium was aspirated, 1mL of serum-free medium containing different concentrations of the antitumor peptide of the present invention (1×ic50,2×ic 50) was added to each well, and incubated in an incubator for 1h. The wells were aspirated, the cells were washed twice with PBS, 1mL of 2.5% glutaraldehyde was added to each well, and the wells were fixed overnight at 4 ℃. Glutaraldehyde was then aspirated and the cells were washed three times with PBS for 5min each. Dehydrating the cells with a series of ethanol (50%, 70%, 80%, 90%, 100%) with each concentration for 15min, and adding 1mL of tertiary solution into each well after dehydrationButanol, standing at room temperature for 2h, sucking out tert-butanol, drying, spraying gold, observing cell morphology with a scanning electron microscope, and adding only blank medium holes as control group. The results are shown in FIG. 3.
As can be seen from fig. 3, the anti-tumor peptide of the present invention can rapidly destroy tumor cell membranes, kill tumor cells, and show concentration dependency of the membrane lysis degree, compared to the blank. The anti-tumor peptide has a unique membrane rupture mechanism, so that the anti-tumor peptide has obvious advantages in resisting multi-drug resistance.
4. PTX administration experiment of paclitaxel combined with chemotherapy
Tumor cells MCF-7 were assayed at 1X 10 per well 4 Individual cells were seeded in 96-well plates and incubated at 37℃and 5% CO 2 Incubate overnight in incubator. Cells were treated with 5% FBS medium containing different concentrations of the antitumor peptide of the present invention and PTX (0.5×IC50 peptide+1. Mu.M PTX,1×IC50 peptide+1. Mu.M PTX) for 24h. Then 10. Mu.L MTT (5 mg/mL) was added per well under dark conditions and incubated for 4h. The medium was gently aspirated, 150 μl DMSO was added to each well and shaken for 10min in a micro-shaker. The absorbance at 490nm was measured using a multifunctional microplate reader and the cell viability was calculated. Cells of the control group were treated with a blank medium without peptide. Experiments were independently repeated three times, four at a time in parallel. The results are shown in FIG. 4.
As can be seen from fig. 4, the combination of the antitumor peptide of the present invention and paclitaxel PTX shows a synergistic effect. Wherein the combination of the antitumor peptide with the low dose PTX (0.5X150+1. Mu.M) exhibits an antitumor effect similar to that of the high dose PTX (20. Mu.M); when the anti-tumor peptide is combined with a low dose PTX (1×IC50+1 mu M), the anti-tumor effect is superior to that of a high dose PTX (20 mu M), the anti-tumor peptide has more excellent anti-tumor activity when being combined with a chemotherapeutic PTX, and the dosage of the chemotherapeutic can be obviously reduced, so that the anti-tumor peptide has therapeutic advantages in combined administration.
Compared with the prior art, the invention has the beneficial effects that:
1. the antitumor peptide has the advantages of simple structure, low molecular weight, novel design, low synthesis cost and is obtained by adopting a conventional solid-phase synthesis method.
2. The antitumor peptide has obvious antitumor activity and low hemolytic activity on tumor cells MCF-7, hela, hepaRG and A549 cells, and has great application prospect in preparing clinical antitumor drugs, combined drugs and resisting multi-drug resistance.
Drawings
FIG. 1 is an experimental diagram of the in vitro anti-tumor activity of the novel high-efficiency low-toxicity anti-tumor peptide of the invention on MCF-7, hela, hepaRG and A549 cells;
FIG. 2 is a diagram of a novel high-efficiency low-toxicity anti-tumor peptide hemolysis experiment;
FIG. 3 is a diagram of a scanning electron microscope experiment of the novel high-efficiency low-toxicity anti-tumor peptide;
FIG. 4 is a graph showing the experimental results of the combination of the novel highly effective and low toxic antitumor peptide and paclitaxel PTX as a chemotherapeutic agent according to the present invention;
FIG. 5 is a mass spectrum of the novel high-efficiency low-toxicity anti-tumor peptide 14L;
FIG. 6 is a mass spectrum of the novel high-efficiency low-toxicity anti-tumor peptide 14D of the invention;
FIG. 7 is a mass spectrum of the novel high-efficiency low-toxicity anti-tumor peptide 14E of the invention;
FIG. 8 is a mass spectrum of the novel high-efficiency low-toxicity anti-tumor peptide 14 Aad;
FIG. 9 is a mass spectrum of the novel high-efficiency low-toxicity antitumor peptide 14 Asd;
FIG. 10 is a mass spectrum of the novel high-efficiency low-toxicity antitumor peptide 14Asu of the invention.
Detailed Description
The synthesis process of the high-efficiency low-toxicity anti-tumor peptide is further described below by specific examples.
Example 1:14L Synthesis
1) Resin activation and pretreatment
0.435g (0.2 mmol) of MBHA resin (load ratio: 0.46 mmol/g) was weighed into a synthesizer, 10mL of DCM was added to swell for 30min, and after three times of washing with DMF, the resin was colorless as judged by ninhydrin chromogenic method, indicating that the resin was normally available.
2) Synthesis of 14L-resin
The Fmoc protecting group was removed from the above-identified resin with 20% piperidine in DMF (v/v) and the resin was blue-violet in color as determined by ninhydrin, indicating removal of the Fmoc protecting group. After four times washing with DMF, fmoc-Leu-OH (0.6 mmol), HBTU (0.6 mmol), HOBT (0.6 mmol) and DIEA (1.2 mmol) were dissolved in DMF and added to the synthesizer and the reaction stirred for 1h. And (3) detecting by using an ninhydrin chromogenic method, wherein the resin is colorless, and Fmoc-Leu-resin is obtained.
According to the method, the subsequent amino acid is condensed and reacted in sequence until Fmoc-Leu-Leu-Lys (Boc) -Lys (Boc) -Leu-Leu-Lys (Boc) -Lys (Boc) -Leu-Leu-Lys (Boc) -Lys (Boc) -Leu-Leu-resin is synthesized, and then Fmoc groups at the tail ends of peptide chains are removed by using a DMF solution containing 20% piperidine to obtain 14L-resin.
3) Polypeptide cleavage
The resulting 14L-resin was washed successively with DCM and MeOH, the resin was drained until it was a powder, and a cleavage agent (TFA: triisoopropylsilane: H was added 2 O=9.5:0.25:0.25 v:v:v peptide chain the resin is cut out from the resin and then, extracting with deionized water and glacial ethyl ether, and freeze-drying to obtain crude peptide freeze-dried powder.
4) Polypeptide purification
Separating and purifying the obtained crude peptide lyophilized powder by RP-HPLC, collecting effluent, lyophilizing again, and identifying by mass spectrum to obtain 14L with molecular weight of 1691Da, wherein the mass spectrum is shown in figure 5, and the amino acid sequence is shown in SEQ ID No. 1.
Example 2:14D Synthesis
1) Resin activation and pretreatment
As in example 1.
2) Synthesis of 14D-resin
The Fmoc protecting group was removed from the above-identified resin with 20% piperidine in DMF (v/v) and the resin was blue-violet in color as determined by ninhydrin, indicating removal of the Fmoc protecting group. After four times washing with DMF Fmoc-Asp (OtBu) -OH (0.6 mmol), HBTU (0.6 mmol), HOBT (0.6 mmol) and DIEA (1.2 mmol) were dissolved in DMF and added to the synthesizer and the reaction stirred for 1h. And (3) detecting by using an ninhydrin chromogenic method, wherein the resin is colorless, and Fmoc-Asp-resin is obtained.
According to the method, the subsequent amino acid is condensed and reacted in sequence until Fmoc-Leu-Leu-Lys (Boc) -Lys (Boc) -Leu-Leu-Lys (Boc) -Lys (Boc) -Leu-Leu-Lys (Boc) -Lys (Boc) -Leu-Asp (OtBu) -resin is synthesized, and then Fmoc groups at the tail ends of peptide chains are removed by using a DMF solution containing 20% piperidine to obtain 14D-resin.
3) Polypeptide cleavage
As in example 1.
4) Polypeptide purification
As in example 1, the molecular weight is 1693Da, the mass spectrum is shown in FIG. 6, and the amino acid sequence is shown in SEQ ID No. 2.
Example 3:14E Synthesis
1) Resin activation and pretreatment
As in example 1.
2) Synthesis of 14E-resin
The Fmoc protecting group was removed from the above-identified resin with 20% piperidine in DMF (v/v) and the resin was blue-violet in color as determined by ninhydrin, indicating removal of the Fmoc protecting group. After four times washing with DMF, fmoc-Glu (OtBu) -OH (0.6 mmol), HBTU (0.6 mmol), HOBT (0.6 mmol) and DIEA (1.2 mmol) were dissolved in DMF and added to the synthesizer and the reaction stirred for 1h. And (3) detecting by using an ninhydrin chromogenic method, wherein the resin is colorless, and Fmoc-Glu-resin is obtained.
According to the method, the subsequent amino acid is condensed and reacted in sequence until Fmoc-Leu-Leu-Lys (Boc) -Lys (Boc) -Leu-Leu-Lys (Boc) -Lys (Boc) -Leu-Leu-Lys (Boc) -Lys (Boc) -Leu-Glu (OtBu) -resin is synthesized, and then Fmoc groups at the tail ends of peptide chains are removed by using a DMF solution containing 20% piperidine to obtain 14E-resin.
3) Polypeptide cleavage
As in example 1.
4) Polypeptide purification
As in example 1, the molecular weight is 1707Da, the mass spectrum is shown in FIG. 7, and the amino acid sequence is shown in SEQ ID No. 3.
Example 4:14Aad synthesis
1) Resin activation and pretreatment
As in example 1.
2) 14Aad-resin synthesis
The Fmoc protecting group was removed from the above-identified resin with 20% piperidine in DMF (v/v) and the resin was blue-violet in color as determined by ninhydrin, indicating removal of the Fmoc protecting group. After four times washing with DMF, fmoc-Aad (OtBu) -OH ((S) -2-Fmoc-aminoadipate 6-t-butyl ester, 0.6 mmol), HBTU (0.6 mmol), HOBT (0.6 mmol) and DIEA (1.2 mmol) were dissolved in DMF and added to the synthesizer and stirred for 1h. And (3) detecting by using an ninhydrin chromogenic method, wherein the resin is colorless, and Fmoc-Aad-resin is obtained.
According to the method, the subsequent amino acid is condensed and reacted in sequence until Fmoc-Leu-Leu-Lys (Boc) -Lys (Boc) -Leu-Leu-Lys (Boc) -Lys (Boc) -Leu-Leu-Lys (Boc) -Lys (Boc) -Leu-Aad (OtBu) -resin is synthesized, and then Fmoc groups at the tail ends of peptide chains are removed by using a DMF solution containing 20% piperidine to obtain 14Aad-resin.
3) Polypeptide cleavage
As in example 1.
4) Polypeptide purification
As in example 1, the molecular weight was 1721Da, and the mass spectrum was as shown in FIG. 8, or: 14Aad, the amino acid sequence of which is Leu-Leu-Lys-Lys-Leu-Leu-Lys-Lys-Leu-Leu-Lys-Lys-Leu-Aad-NH 2 。
Example 5:14Asd synthesis
1) Resin activation and pretreatment
As in example 1.
2) 14Asd-resin synthesis
The Fmoc protecting group was removed from the above-identified resin with 20% piperidine in DMF (v/v) and the resin was blue-violet in color as determined by ninhydrin, indicating removal of the Fmoc protecting group. After four times washing with DMF Fmoc-Asd (OtBu) -OH ((S) -2-Fmoc-aminopimelic acid-7-tert-butyl ester, 0.6 mmol), HBTU (0.6 mmol), HOBT (0.6 mmol) and DIEA (1.2 mmol) were dissolved in DMF and added to the synthesizer and stirred for 1h. And (3) detecting by using an ninhydrin chromogenic method, wherein the resin is colorless, and Fmoc-Asd-resin is obtained.
According to the method, the subsequent amino acid is condensed and reacted in sequence until Fmoc-Leu-Lys (Boc) -Lys (Boc) -Leu-Leu-Lys (Boc) -Lys (Boc) -Leu-Leu-Lys (Boc) -Lys (Boc) -Leu-Asd (OtBu) -resin is synthesized, and then Fmoc groups at the tail ends of peptide chains are removed by using a DMF solution containing 20% piperidine to obtain 14Asd-resin.
3) Polypeptide cleavage
As in example 1.
4) Polypeptide purification
As in example 1, the molecular weight was 1735Da, the mass spectrum was shown in FIG. 9, and the amino acid sequence was Leu-Leu-Lys-Lys-Leu-Leu-Lys-Lys-Leu-Leu-Asd-NH 2 。
Example 6: synthesis of 14Asu
1) Resin activation and pretreatment
As in example 1.
2) Synthesis of 14Asu-resin
The Fmoc protecting group was removed from the above-identified resin with 20% piperidine in DMF (v/v) and the resin was blue-violet in color as determined by ninhydrin, indicating removal of the Fmoc protecting group. After four times washing with DMF, fmoc-Asu (OtBu) -OH ((S) -2-Fmoc-aminosuberic acid-8-tert-butyl ester, 0.6 mmol), HBTU (0.6 mmol), HOBT (0.6 mmol) and DIEA (1.2 mmol) were dissolved in DMF and added to the synthesizer and stirred for 1h. And (3) detecting by using an ninhydrin chromogenic method, wherein the resin is colorless, and Fmoc-Asu-resin is obtained.
According to the method, the subsequent amino acid is condensed and reacted in sequence until Fmoc-Leu-Lys (Boc) -Lys (Boc) -Leu-Leu-Lys (Boc) -Lys (Boc) -Leu-Leu-Lys (Boc) -Lys (Boc) -Leu-Asu (OtBu) -resin is synthesized, and then Fmoc groups at the tail ends of peptide chains are removed by using a DMF solution containing 20% piperidine to obtain 14Asu-resin.
3) Polypeptide cleavage
As in example 1.
4) Polypeptide purification
As in example 1, the molecular weight was 1749Da, the mass spectrum was as shown in FIG. 10, and the amino acid sequence was Leu-Leu-Lys-Lys-Leu-Leu-Lys-Lys-Leu-Leu-Asu-NH 2 。

Claims (4)

1. A high-efficiency low-toxicity antitumor peptide rich in leucine and lysine is characterized in that the antitumor peptide has a structural general formula:
LLKKLLKKLLKKLX-NH 2 marked 14X;
wherein x= L, D, E, aad, asd, asu, aad is 2-aminoadipic acid, asd is aminopimelic acid, asu is aminosuberic acid.
2. The leucine and lysine rich high-efficiency low-toxicity antitumor peptide according to claim 1, wherein said antitumor peptide is:
14L, the amino acid sequence of which is shown as SEQ ID No. 1;
or: 14D, the amino acid sequence of which is shown as SEQ ID No. 2;
or: 14E, the amino acid sequence of which is shown in SEQ ID No. 3;
or: 14Aad, the amino acid sequence of which is Leu-Leu-Lys-Lys-Leu-Leu-Lys-Lys-Leu-Leu-Lys-Lys-Leu-Aad-NH 2 ；
Or: 14Asd, the amino acid sequence of which is Leu-Leu-Lys-Lys-Leu-Leu-Lys-Lys-Leu-Leu-Lys-Lys-Leu-Asd-NH 2 ；
Or: 14Asu, the amino acid sequence of which is Leu-Leu-Lys-Lys-Leu-Leu-Lys-Lys-Leu-Leu-Lys-Lys-Leu-Asu-NH 2 。
3. The use of a class of highly potent low toxic antitumor peptides enriched in leucine and lysine according to claim 1 or 2 for the preparation of clinical antitumor drugs.
4. The use of a class of highly potent low toxic antitumor peptides enriched in leucine and lysine according to claim 3 for the preparation of clinical antitumor drugs, wherein the antitumor peptide is combined with paclitaxel PTX to increase antitumor activity and reduce the dosage of paclitaxel PTX.
CN202310945080.7A 2023-07-28 2023-07-28 Leucine and lysine-rich high-efficiency low-toxicity anti-tumor peptide and application thereof Pending CN117343142A (en)

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