CN105622722B

CN105622722B - Peptide variant XA3 for treating cancer and application thereof

Info

Publication number: CN105622722B
Application number: CN201610002817.1A
Authority: CN
Inventors: 朱凌玮
Original assignee: Beijing Oriental Medicine Polytron Technologies Inc
Current assignee: Beijing Oriental Medicine Polytron Technologies Inc
Priority date: 2014-05-29
Filing date: 2014-05-29
Publication date: 2019-12-24
Anticipated expiration: 2034-05-29
Also published as: CN105481949A; CN105601712A; CN105601714B; CN105601714A; CN105601710A; CN105504016A; CN105597078B; CN105601715B; CN105585615A; CN105601710B; CN105601711A; CN105601711B; CN105585615B; CN105601713A; CN105622722A; CN105597078A; CN105481948B; CN105481948A; CN105504017B; CN105504017A

Abstract

The present invention provides a series of variants of anti-cancer peptides of SEQ ID No:2-15 and pharmaceutical use thereof. These peptide variants are represented in the sequence SEQ ID No:1, the polypeptide is obtained by a series of point mutations and specific modification at two ends respectively, and compared with a wild type, the peptide variants have stronger effect of killing tumor cells and have wide pharmaceutical prospect.

Description

Peptide variant XA3 for treating cancer and application thereof

Technical Field

The present invention is in the field of biotechnology, and in particular, the present invention relates to peptide variants for the treatment of cancer.

Background

Microtubules are a constituent of one of the three filamentous networks of the cytoskeleton, complex, unstable polymers, the basic units of microtubules being α -and β -tubulin dimers. For example, they are responsible for a variety of cellular movements, axonal transport, or motility, and in particular, they provide the basic material for the formation of mitotic spindles. The microtubes are continually reformed into hollow tubes of different lengths. Thus, by disrupting the microtubule dynamics of cancer cells, the peptides of the invention can act as antimitotic agents that prevent cancer cell proliferation (slow or prevent cancer cell mitosis).

The active peptides of the invention also appear to specifically alter the plasma membrane of cancer cells and appear not to cause these same alterations in healthy cells of the host organism. The active peptides have polycationic properties, so that they act on the plasma membrane of cancer cells that are rich in negative charges.

In the prior art, it is common practice to specifically mutate and substitute the active site of a protein or peptide in order to increase the activity of the protein or peptide, and thereby search for more active variants.

Disclosure of Invention

The peptides shown in SEQ ID No. 1 are known in the art and can be used to treat cancer. But their activity is not very satisfactory. In order to further improve the biological activity of the peptide, the applicant carries out point mutation on a specific site in the peptide shown in SEQ ID No. 1 and carries out specific modification on two ends respectively through a large number of experiments, thereby obtaining a variant with higher cancer inhibition activity than the peptide shown in SEQ ID No. 1.

Specifically, the sequence of the peptide used as an active ingredient for treating cancer is shown below (table I):

SEQ ID NO (XA code)	Sequence of	Number of amino acids
			1（XA1）	INWLKIAKKVAGML-NH₂	14
2（XA2）	SINWLKIAKKVAGML-NH₂	15
			3（XA3）	SINWLAIAKKVAGML-NH₂	15
4（XA4）	SINWLAIAAKVAGML-NH₂	15
			5（XA5）	SINWLAIAAKVGGML-NH₂	15
6（XA6）	SINWLKIAKKVAGMLL-NH₂	16
			7（XA7）	SINWLKIAKKVAGMLL-NH₂	16
8（XA8）	SINWLAIAKKVAGMLL-NH₂	16
			9（XA9）	SINWLAIAAKVAGMLL-NH₂	16
10（XA10）	SINWLAIAAKVGGMLL-NH₂	16
			11（XA11）	INWLINWLKIAKKVAGML-NH₂	18
12（XA12）	INWLSINWLKIAKKVAGML-NH₂	19
			13（XA13）	INWLSINWLAIAKKVAGML-NH₂	19
14（XA14）	INWLSINWLAIAAKVGGMLL-NH₂	20
			15（XA15）	INWLSINWLKIAKKVAGMLL-NH₂	20
16（XA16）	INWL INWLAIAKKVAGML-NH₂	18
			17（XA17）	INWLSINWLAIAKKVAGMLL-NH₂	20

The Applicant's studies of the peptides SEQ ID No. 2 to SEQ ID No. 15 have demonstrated that these peptides have a higher cytotoxic activity towards cancer cells compared to the peptide SEQ ID No. 1, while having very little or No toxicity towards the cells of the host to be treated. The active peptides of the invention specifically alter the microtubules of cancer cells.

The peptides cited in table I are used as drugs for treating cancer, and in particular, are effective in treating leukemia, liver cancer, glioblastoma, and the like.

According to another characteristic of the invention, the peptides of sequences SEQ ID No. 1 to SEQ ID No. 15 are produced by chemical synthesis.

Another object of the invention relates to a pharmaceutical composition comprising one of the peptides defined above.

Advantageously, the composition is in the form of an aqueous suspension or solution, or in the form of an uncoated or coated tablet, such as a pill, gel capsule, capsule or powder, in the dry state.

If the pharmaceutical composition is in a dry state, it may be mixed with one or more inert diluents such as starch, cellulose, sucrose, lactose, or silicon dioxide, and the like. It may also contain other substances in the dry state, for example one or more lubricants such as magnesium stearate or talc, colorants, coatings (sugar-coated tablets) or varnishes.

If the pharmaceutical composition of the present invention is in liquid form, it may include pharmaceutically acceptable solutions, suspensions, emulsions, syrups and elixirs containing inert diluents such as water, ethanol, glycerol, vegetable oils or liquid paraffin. The pharmaceutical composition may also comprise other liquid substances than diluents, such as wetting agents, sweeteners, thickeners, flavoring agents or stabilizer products.

Said composition, optionally in combination with a pharmaceutically acceptable non-toxic inert excipient or vehicle, characterized in that it can be applied topically, transdermally or transdermally; orally taking; parenterally; the composition is administered topically in the form of nasal or bronchial application.

Pharmaceutical compositions for parenteral administration may be aqueous or non-aqueous solutions, suspensions or emulsions. These formulations are prepared from water, propylene glycol, vegetable oils, especially olive oil and sesame oil, injectable organic esters such as ethyl oleate, or other suitable organic solvents as solvents or vehicles. The pharmaceutical compositions may also contain adjuvants, in particular wetting, isotonizing, emulsifying, dispersing and stabilizing agents.

For topical application, the medicament is advantageously administered in the form of an ointment, cream, gel or patch.

In addition, the pharmaceutical compositions may be used alone or in combination with other drugs in a given treatment, or in combination with radiation therapy or surgery.

Detailed Description

A)Synthesis of peptides

Synthesis and purification of peptides SEQ ID No. 1 to SEQ ID No. 17

The peptides were prepared by manual isolation of solid phase synthesis using N-9-fluorophenylmethoxycarbonyl and Novasyn TGS resin. Side chain protecting groups include the tert-butyl group of serine and the tert-butoxycarbonyl group of lysine. To prepare a peptide with an acetylated N-terminal residue, acetic anhydride was added to the resin-peptide complex after the last amino acid residue was attached, and stirring was maintained for 1 hour to promote acetylation.

The resin-peptide complex was cleaved by treatment with trifluoroacetic acid/1, 2-ethanedithiol/anisole/phenol/water (volume ratio 82.5:2.5:5:5: 5) at a concentration of 10mL. 1 for 2 hours at room temperature. Followed by filtration to remove the resin. The crude peptide was then precipitated by addition of 4 ℃ diethyl ether and collected after centrifugation at 1000g for 15 minutes at room temperature. The crude peptide was subsequently dissolved in water and chromatographed by RP-HPLC using a Shiseido C-18 semi-preparative column (250 mm. times.10.0 mm; 5 μm) eluting with 40% (v/v) acetonitrile containing 0.1% (v/v) TFA at a rate of 2mL/min with a constant solvent. Elution was adjusted at 215nm with a UV-DAD detector (ultraviolet diode array detector) (Shimadzu, model SPD-M10A), and each eluted fraction was manually collected in a 2mL glass vial. The sequence of the synthesized peptides (both acetylated and non-acetylated peptides) was determined by HPLC and ESI-MS analysis. After the peptides were synthesized, purified and tested, the peptide sequences shown in table I were confirmed.

B)Activity of peptides SEQ ID Nos 1-17 on healthy cells

Two non-tumorigenic fibroblasts, FIBRO1 and FIBRO2, were contacted with the peptide SEQ ID nos 1-17 to see if the microtubule network of healthy cells was also disrupted by the peptide. In the case of using a peptide concentration of 15. mu.M, human fibroblasts representing a variety of normal cells did not respond at all to the peptides SEQ ID Nos: 1-15 (No change in polymerized tubulin density). Whereas the fibroblasts treated with SEQ ID Nos 16-17 had a greater effect on tubulin density, with a thickness reduction of about 15%. This indicates that the peptides SEQ ID Nos 1-15 are inactive against healthy cells. When the peptides SEQ ID Nos 1-15 are used as therapeutic agents in mammals, especially humans, there is a high probability that few side effects will actually occur.

C) Determination of cell proliferation and cell viability

The following cell lines (Table II) were analyzed to determine and quantify the activity of the peptides SEQ ID Nos 1-17.

TABLE II

Tissue type	Cell lines
		Acute promyelocytic leukemia cell line	NB4
Liver cancer	HepG2
		Glioblastoma	U87M6

Approximately 10000 cells were seeded in 96-well microplates at a volume of 0.2mL per well and after 24 hours, treated with the peptide SEQ ID Nos 1-17. Cells were exposed to CO in the presence of the peptide SEQ ID Nos 1-17₂Incubate at 37 ℃ for 48 hours in an incubator. After incubation, MTT reagent or (3- (4, 5-dimethyl-2-yl) -2, 5-diphenyltetrazolium bromide (0.5 mg/ml in a volume of 0.1 ml) was added to each well and incubated at 37 ℃ for 2 hours.Wells without peptide were used to confirm the viability of the control cells. The value of IC50 (concentration of peptide SEQ ID Nos: 1-17 causing 50% cytotoxicity on these cell lines), μ M, was evaluated for each cell by the ratio of the absorbance of the treated cells to the absorbance of the control cells.

TABLE III

SEQ ID NO (XA code)	NB4（IC50）	HepG2（IC50）	U87M6（IC50）
				1（XA1）	52.2	35.8	15.41
2（XA2）	37.3	29.3	10.9
				3（XA3）	32.5	27.4	9.8
4（XA4）	34.6	25.4	10.3
				5（XA5）	31.9	26.9	10.1
6（XA6）	40.1	24.8	9.7
				7（XA7）	34.9	28.7	9.6
8（XA8）	35.2	27.3	10.0
				9（XA9）	37.8	28.1	10.2
10（XA10）	36.3	25.1	10.4
				11（XA11）	30.9	20.9	9.9
12（XA12）	29.8	19.9	9.3
				13（XA13）	31.7	21.7	9.7
14（XA14）	39.5	22.5	9.7
				15（XA15）	28.7	20.1	10.1
16（XA16）	60.9	45.9	30.5
				17（XA17）	65.3	50.5	35.7

As shown in Table III, peptides SEQ ID Nos. 2-15 significantly increased the proliferation of the above three tumor cells. Compared with the peptide SEQ ID No. 1, the dosage of the polypeptide with toxicity to cancer cells is obviously reduced, and the peptide SEQ ID No. 16 and the peptide SEQ ID No. 17 improve the IC50 value, so that the peptide SEQ ID No. 2-15 has better inhibition effect on the tumor cells compared with the prior art, and can be further used for the related treatment of tumors.

Resistance of cancer cells to peptides

1-15 of SEQ ID No. are respectively used for carrying out drug resistance experiments on cancer cells, the experimental method is a conventional method in the field, and the result shows that the peptide has a rapid drug resistance process without induction.

Sequence listing

<110> Relinanwei

< 120 > a peptide variant XA3 for treating cancer and use thereof

〈160〉17

〈210〉1

〈211〉14

〈212〉PRT

〈213〉artificial sequence

〈400〉1

INWLKIAKKVAGML

〈210〉2

〈211〉14

〈212〉PRT

〈213〉artificial sequence

〈400〉2

SINWLKIAKKVAGML

〈210〉3

〈211〉15

〈212〉PRT

〈213〉artificial sequence

〈400〉3

SINWLAIAKKVAGML

〈210〉4

〈211〉15

〈212〉PRT

〈213〉artificial sequence

〈400〉4

SINWLAIAAKVAGML

〈210〉5

〈211〉15

〈212〉PRT

〈213〉artificial sequence

〈400〉5

SINWLAIAAKVGGML

〈210〉6

〈211〉16

〈212〉PRT

〈213〉artificial sequence

〈400〉6

SINWLKIAKKVAGMLL

〈210〉7

〈211〉16

〈212〉PRT

〈213〉artificial sequence

〈400〉7

SINWLKIAKKVAGMLL

〈210〉8

〈211〉16

〈212〉PRT

〈213〉artificial sequence

〈400〉8

SINWLAIAKKVAGMLL

〈210〉9

〈211〉16

〈212〉PRT

〈213〉artificial sequence

〈400〉9

SINWLAIAAKVAGMLL

〈210〉10

〈211〉16

〈212〉PRT

〈213〉artificial sequence

〈400〉10

SINWLAIAAKVGGMLL

〈210〉11

〈211〉18

〈212〉PRT

〈213〉artificial sequence

〈400〉12

INWLINWLKIAKKVAGML

〈210〉12

〈211〉19

〈212〉PRT

〈213〉artificial sequence

〈400〉12

INWLSINWLKIAKKVAGML

〈210〉13

〈211〉19

〈212〉PRT

〈213〉artificial sequence

〈400〉13

INWLSINWLAIAKKVAGML

〈210〉14

〈211〉20

〈212〉PRT

〈213〉artificial sequence

〈400〉14

INWLSINWLAIAAKVGGMLL

〈210〉15

〈211〉20

〈212〉PRT

〈213〉artificial sequence

〈400〉15

INWLSINWLKIAKKVAGMLL

〈210〉16

〈211〉18

〈212〉PRT

〈213〉artificial sequence

〈400〉16

INWLINWLAIAKKVAGML

〈210〉17

〈211〉20

〈212〉PRT

〈213〉artificial sequence

〈400〉17

INWLSINWLAIAKKVAGMLL

Claims

1. A peptide for use in the treatment of cancer, characterized by: XA3, and the amino acid sequence is SEQ ID No. 3.

2. Use of a peptide according to claim 1 for the preparation of a medicament for the treatment of leukemia, liver cancer or glioblastoma.