Antiradiation polypeptide and application thereof

Abstract

The application discloses an anti-radiation polypeptide and application thereof, which is obtained by cyclization of TP508 at the head and tail, and the amino acid sequence of TP508 is AGYKPDEGKRGDACEGDSGGPFV. The application mainly modifies TP508 to obtain a polypeptide drug with stronger anti-radiation effect and better drug property. The polypeptide can prolong the survival period of mice after radiation, can be applied to alleviate radiation damage treatment, and provides a new scheme and new idea for radiation treatment.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and provides an anti-radiation polypeptide and application thereof. BACKGROUND

[0002] Radiation protection agents are drugs used for preventing, reducing or treating ionizing radiation damage. According to the time of use, they are divided into radiation protection agents, radiation mitigating agents and radiation therapeutic agents. The core mechanism of radiation protection agents is to inhibit cell or tissue damage caused by radiation, enhance the resistance of organisms to radiation exposure, and promote the repair of radiation-induced tissue damage. Currently marketed radiation protection agents include amifostine, palifosine, filgrastim, pegfilgrastim, sargramostim, radionuclide elimination agents, etc. Radiation protection agents undergoing clinical trials include elamipretary, HemaMax TM , pharmacological dose ascorbic acid, entomod, genistein, beclometasone dipropionate, etc. These radiation protection agents can inhibit radiation-induced damage to some extent. However, some radiation protection agents have protection limitations, potential toxic side effects, high prices and strict use conditions, which suggest the need to develop new anti-radiation drugs to achieve more efficient, safe, economical and convenient radiation protection solutions to meet the higher demands of clinical practice and emergency response.

[0003] The closest prior art to the present application is the peptide TP508 (AGYKPDEGKRGDACEGDSGGPFV) (Kantara, C., et al., Novel regenerative peptide TP508 mitigates radiation-induced gastrointestinal damage by activating stem cells and preserving crypt integrity. Lab Invest, 2015. 95(11): p. 1222-33.). TP508 is a 23-amino-acid non-proteolytic thrombin peptide, representing a part of the thrombin molecule receptor binding region. TP508 can reduce the effects of radiation on human endothelial cells, restore endothelial NO production, vascular formation and accelerate repair of radiation-induced DNA double-strand breaks.

[0004] The present application mainly modifies TP508 to obtain a polypeptide drug with stronger anti-radiation effect and better drug properties. SUMMARY

[0005] Therefore, the purpose of the present application is to provide an anti-radiation polypeptide and application thereof to prolong the growth period of irradiated mice.

[0006] To achieve the above object, the present application provides the following technical solutions.

[0007] 1. An anti-radiation polypeptide, which is a cyclic peptide obtained by cyclization of TP508, the amino acid sequence of TP508 being AGYKPDEGKRGDACEGDSGGPFV, as shown in SEQ ID NO. 1.

[0008] 2. A preparation method of the anti-radiation polypeptide, which comprises differentiating protection of an α-amino group at an N-terminal amino acid of TP508 and removal of the α-amino group, and then condensing the α-amino group with a carboxyl group at a C-terminal of a polypeptide to obtain a cyclic peptide.

[0009] As one of the preferred technical solutions, the Fmoc solid-phase synthesis method is adopted.

[0010] As one of the further preferred technical solutions, the first amino acid at the C-terminal of the polypeptide is first connected to 2-CTC-resin which is sensitive to weak acid, and then amino acids are sequentially condensed according to the sequence of the polypeptide from the C-terminal to the N-terminal; the amino acid is protected by a Fmoc (9-fluorenylmethyloxycarbonyl) group at the amino-terminal end, and is coupled with a growing chain (a polypeptide chain gradually constructed on a resin carrier; amino acid residues are gradually added from the C-terminal (carboxyl-terminal) to the N-terminal (amino-terminal) until the desired sequence length is reached) after being activated at the carboxylic acid terminal; then the Fmoc group is removed by piperidine treatment and the process is repeated.

[0011] As one of the still further preferred technical solutions, after the peptide assembly is completed, the peptide can be removed from the resin by treatment with a 2% trifluoroacetic acid (TFA) aqueous solution; at this time, the protection groups on the side chains of the amino acids are retained, only the amino group at the N-terminal and the carboxyl group at the C-terminal of the polypeptide are exposed, and a crude fully-protected linear peptide is obtained; after the crude fully-protected linear peptide is dissolved, the exposed amino group at the N-terminal and the carboxyl group at the C-terminal of the polypeptide are condensed using a condensing agent to obtain a fully-protected cyclic peptide, and then a 95% TFA aqueous solution is used to remove all the side chain protection groups of the obtained fully-protected cyclic peptide, to obtain the target cyclic peptide. The polypeptide is purified by high performance liquid chromatography (HPLC) to remove by-products and unreacted substances.

[0012] HPLC chromatographic conditions:

[0013] Chromatographic column: kromasil C18-5 column (4.6*150mm); mobile phase A is a 0.1% trifluoroacetic acid acetonitrile solution, B is a 0.1% trifluoroacetic acid aqueous solution, gradient elution (at 0.01 min, 5% A 95% B, at 25.0 min, 75% A 25% B, at 30 min, 90% A 10% B; the change of the mobile phase is in volume percent); flow rate 1.0 mL / min; detection wavelength 214 nm; injection volume 20 ul.

[0014] MS Mass Spectrometry Conditions: Expected MS 2294.47; flow rate 0.2 mL / min; run time 1 min; buffer A is 0.1% formic acid in water, buffer B is 0.1% formic acid in acetonitrile.

[0015] 3. Use of the aforementioned anti-radiation polypeptide in the preparation of an anti-radiation drug.

[0016] The beneficial effects of the present application are:

[0017] The closest prior art to the present application is the peptide TP508 (AGYKPDEGKRGDACEGDSGGPFV) (Kantara, C., et al., Novel regenerative peptide TP508 mitigates radiation-induced gastrointestinal damage by activating stem cells and preserving crypt integrity. Lab Invest, 2015. 95(11): p. 1222-33.). TP508 is a 23-amino acid non-proteolytic thrombin peptide, representing a portion of the thrombin molecule receptor binding region. TP508 reduces the effects of radiation on human endothelial cells, restores endothelial NO production, vascular formation and accelerates repair of radiation-induced DNA double-strand breaks.

[0018] The present application mainly modifies TP508 to obtain a polypeptide drug with stronger anti-radiation effect and better drugability. The polypeptide can prolong the survival of mice after radiation and can be applied to alleviate radiation damage treatment, providing a new scheme and new idea for radiation treatment.

[0019] Other advantages, objects, and features of the present application will be set forth in part in the following specification taken in conjunction with the accompanying drawings, and in part will become apparent to those skilled in the art from a consideration of the following specification and drawings, or can be learned from the practice of the present application. The objects and other advantages of the present application can be realized and attained by means of the instrumentalities and combinations pointed out in the following specification. BRIEF DESCRIPTION OF DRAWINGS

[0020] In order to make the objects, technical solutions and advantages of the present application clearer, the preferred detailed description of the present application will be combined with the drawings to describe the present application, in which:

[0021] Figure 1 Anti-N peptide HPLC analysis results;

[0022] Figure 2 Anti-N peptide MS analysis results;

[0023] Figure 3 Survival curve of Balbc mice after Anti-N peptide administration;

[0024] Figure 4 Survival curve of Balbc mice after Anti-N peptide administration;

[0025] Figure 5 Survival curve of Balbc mice after Anti-N peptide administration;

[0026] Figure 6 Survival curve of Balbc mice after Anti-N peptide administration;

[0027] Figure 7 Structure of Anti-N peptide peptide fragment. DETAILED DESCRIPTION

[0028] The application will be further described below in conjunction with the specific embodiments.

[0029] Unless otherwise indicated, technical or conditions not specified in the examples were carried out according to the techniques or conditions described in the literature or according to the product instructions. Unless otherwise indicated, the reagents or instruments used were conventional products that can be purchased through regular channels.

[0030] Example 1:

[0031] Preparation of Anti-N peptide

[0032] TP508 (AGYKPDEGKRGDACEGDSGGPFV) was acylated at both ends to form a ring. Figure 7

[0033] Using the method of Fmoc solid-phase synthesis, the first amino acid at the C-terminus of the polypeptide was connected to 2-CTC-resin which is sensitive to weak acid, and then according to the sequence of the polypeptide, the amino acids were condensed in order from C-terminus to N-terminus. The amino acid at its amino terminus was protected by Fmoc (9-fluorenylmethoxycarbonyl) group, and after the carboxylic acid terminus was activated, it was coupled with the growing chain. Then the Fmoc group was removed by piperidine treatment and the process was repeated.

[0034] ​After the peptide assembly is completed, it can be removed from the resin by treating with a 2% trifluoroacetic acid (TFA) aqueous solution; at this time, the protecting groups on the amino acid side chains are retained, only exposing the amino group at the N-terminus and the carboxyl group at the C-terminus of the polypeptide, to obtain a crude fully-protected linear peptide; after the crude fully-protected linear peptide is dissolved, the exposed N-terminal amino group and C-terminal carboxyl group of the polypeptide are condensed using a condensing agent to form a fully-protected cyclic peptide, and then a 95% TFA aqueous solution is used to remove all the side chain protecting groups of the obtained fully-protected cyclic peptide, to obtain the target cyclic peptide. The polypeptide is purified by high performance liquid chromatography (HPLC) to remove byproducts and unreacted substances (such as Figure 1 as shown).

[0035] HPLC chromatographic conditions:

[0036] Chromatographic column: kromasil C18-5 column (4.6*150mm); mobile phase A is a 0.1% trifluoroacetic acid acetonitrile solution, B is a 0.1% trifluoroacetic acid aqueous solution, gradient elution (at 0.01 min, 5% A 95% B, at 25.0 min, 75% A 25% B, at 30 min, 90% A 10% B; all changes in mobile phase are in volume percent); flow rate 1.0 mL / min; detection wavelength 214 nm; injection volume 20ul.

[0037] The molecular weight and composition of the polypeptide are determined by mass spectrometry (MS) (such as Figure 2 as shown), MS mass spectrometry conditions: expected MS is 2294.47; flow rate 0.2 mL / min; run time 1 min; buffer A is a 0.1% formic acid aqueous solution, buffer B is a 0.1% formic acid acetonitrile solution.

[0038] Example 2:

[0039] Polypeptide treatment test in mice

[0040] Take 10 to 12 weeks old Balbc male mice (Chongqing Tengxin Bill Experimental Animal Sales Co., Ltd.), irradiate with an X-ray irradiator, the dose is 6Gy (1.252Gy / min), and observe the survival time of the mice after administration.

[0041] Experiment one: mice were randomly assigned to four treatment groups (15 mice per group): saline treatment group (Ctrl), TP508 treatment group, radiation peptide (Anti-N) treatment group and radiation peptide (802) treatment group. One hour after irradiation, the mice were injected with 0.035mg / ml polypeptide (the control group was injected with saline), and the survival time of the mice was observed, and the results are as follows Figure 3 , Figure 4As shown, the modified anti-radiation polypeptide (Anti-N) can prolong the survival of mice. The amino acid sequence of the radiation peptide (802) is AGYKPDEGK(gamma-Glu-C16)RGDACEGSDGGPFV-NH2, and the amino acid sequence of TP508 is

[0042] AGYKPDEGKRGDACEGDSGGPFV.

[0043] Experiment two: In order to further compare the effects of TP508 and Anti-N, the irradiation experiment was performed again. The mice were randomly assigned to three treatment groups (15 mice in each group): saline treatment group (Ctrl), TP508 treatment group and anti-radiation peptide (Anti-N). One hour after irradiation, the mice were injected with 0.05 mg / ml polypeptide (the control group was injected with saline), and the survival of the mice was observed, and the results are shown in Figure 5 、 Figure 6

[0044] Balbc male mice were administered 1 hour after 6Gy X-ray irradiation (n=15 in each group), group 1 was treated with saline, group 2 was treated with TP508, and group 3 was treated with the optimized anti-radiation peptide (Anti-N). After the mice were injected with 0.05 mg / ml polypeptide (the control group was injected with saline), the death date Figure 5 ) and survival Figure 6 ) of the mice were observed. The results show that the modified anti-radiation polypeptide (Anti-N) can prolong the survival of mice, and the effect is better than that of TP508.

[0045] Finally, it should be pointed out that the above examples are only used to illustrate the technical solutions of the present application and are not limiting. Although the present application has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solutions of the present application can be modified or replaced by equivalents without departing from the spirit and scope of the present technical solutions, and they should be covered in the scope of the claims of the present application.​

Claims

1. An anti-radiation polypeptide, characterized in that, is obtained by cyclization of TP508 at both ends, and the amino acid sequence of TP508 is AGYKPDEGKRGDACEGDSGGPFV, as shown in SEQ ID NO.

1.

2. The preparation method of the anti-radiation polypeptide according to claim 1, wherein the N-terminal amino acid of TP508 is differentially protected and removed, and then the alpha-amino group is condensed with the C-terminal carboxyl group of the polypeptide to realize the cyclization of the polypeptide at both ends.

3. The production method according to claim 2, characterized by, Fmoc solid phase synthesis is adopted.

4. The production method according to claim 3, characterized by, First, the first amino acid at the C-terminal of the polypeptide is connected to 2-CTC resin which is sensitive to weak acid, and then according to the sequence of the polypeptide, the amino acids are sequentially condensed from the C-terminal to the N-terminal; the amino acid is protected by Fmoc group at the amino terminal, and after the carboxylic acid terminal is activated, it is coupled with the growing chain; then the Fmoc group is removed by piperidine treatment and the process is repeated.

5. The production method according to claim 4, characterized by, After the peptide assembly is completed, it is removed from the resin by treating with 2% trifluoroacetic acid aqueous solution; at this time, the protection group on the side chain of the amino acid is retained, only the amino group at the N-terminal and the carboxyl group at the C-terminal of the polypeptide are exposed, and a crude fully protected linear peptide is obtained; after the crude fully protected linear peptide is dissolved, the condensing agent is used to condense the exposed amino group at the N-terminal and the carboxyl group at the C-terminal of the polypeptide into a fully protected cyclic peptide, and then a 95% TFA aqueous solution is used to remove all the side chain protection groups of the obtained fully protected cyclic peptide to obtain the target cyclic peptide.

6. The use of the anti-radiation polypeptide according to claim 1 in the preparation of anti-radiation drugs.

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