Cyclic heptapeptide as well as preparation method and application thereof

Abstract

The invention relates to cyclic heptapeptide as well as a preparation method and application thereof. Specifically, the invention provides a cyclic peptide, and the cyclic peptide is characterized in that the cyclic heptapeptide is a cyclic peptide composed of an amino acid sequence as shown in SEQ ID NO. 1: Thr-Tyr-Val-Pro-Lys-Ala-Phe. The cyclic heptapeptide disclosed by the invention has the characteristics of stable structure, high biological activity, good pharmacokinetic characteristics, strong targeting specificity and the like, so that the cyclic heptapeptide has more remarkable advantages in drug development and biomedical application.

Description

Technical Field

[0001] The present invention relates to the field of biopharmaceutical technology, and more specifically, to a cyclic heptapeptide and a preparation method and application thereof. Background Art

[0002] Straight-chain peptide (Thr-Tyr-Val-Pro-Lys-Ala-Phe) (abbreviated as TYVPKAF) has therapeutic effects in osteoarthritis, but it does not have advantages in terms of biological activity and targeting specificity.

[0003] Cyclic peptides are one of the most attractive molecules in modern drug discovery due to their small molecular weight, high affinity and specificity, and ease of synthesis. Compared to linear peptides, the conformational constraints imposed by cyclization can make cyclic peptides highly resistant to proteolysis by endogenous proteases.

[0004] Furthermore, the rigidity of cyclic peptides can improve their pharmacokinetic and pharmacodynamic properties with respect to absorption and have a significant impact on their ability to passively permeate membranes to reach intracellular targets.

[0005] Therefore, there is an urgent need to find a modification method with high biological activity and strong targeting specificity to transform the linear peptide (TYVPKAF) into a cyclic peptide, so as to give it more significant advantages in drug development and biomedical applications. Summary of the Invention

[0006] The purpose of the present invention is to provide a cyclic peptide with stable structure, high biological activity, good pharmacokinetic properties and strong targeting specificity, so as to make it have more significant advantages in drug development and biomedical applications.

[0007] In a first aspect of the present invention, a cyclic peptide is provided, wherein the cyclic peptide is a cyclic peptide consisting of an amino acid sequence as shown in SEQ ID NO.1: Thr-Tyr-Val-Pro-Lys-Ala-Phe, and the structural formula of the cyclic peptide is:

[0008] .

[0009] In another preferred embodiment, the cyclization mode of the cyclic peptide is that the threonine at position 1 and the phenylalanine at position 7 of the linear peptide form a peptide bond through dehydration condensation, thereby forming a cyclic structure.

[0010] In the second aspect of the present invention, a gene sequence encoding the cyclic peptide described in the first aspect of the present invention is provided.

[0011] In the third aspect of the present invention, a fusion protein containing the cyclic peptide described in the first aspect of the present invention is provided.

[0012] In a fourth aspect of the present invention, a method for preparing the cyclic peptide according to the first aspect of the present invention is provided, wherein the method comprises solid phase synthesis.

[0013] In another preferred embodiment, the method comprises the following steps:

[0014] (1) Solid-phase synthesis stage: Select the resin carrier 2-chlorotrityl chloride resin (2-CTC RESIN), and form an ester bond between the chlorine atom and the carboxyl group of phenylalanine (Phe) to anchor the amino acid on the resin; and add the side chain protecting group Fmoc after each coupling reaction to protect the amino group of Phe with Fmoc to obtain the synthetic compound Fmoc-Phe-2-ctc-Resin;

[0015] (2) Fmoc removal: Use piperidine / DMF solution to remove the Fmoc group, exposing the amino group of Phe to obtain the resin complex NH2-Phe-2-ctc-Resin with free amino group;

[0016] (3) Connecting Fmoc-Ala-OH: Activate the carboxyl group of Fmoc-protected alanine (Fmoc-Ala-OH) and condense it with the amino group of the Phe exposed on the resin in step (2) to form a peptide bond to obtain Fmoc-Ala-Phe-2-ctc Resin;

[0017] (4) After sequentially connecting the protected amino acids, the resin is cleaved and the amino and carboxyl groups are subjected to dehydration condensation to obtain the cyclic peptide described in the first aspect of the present invention.

[0018] In a fifth aspect, the present invention provides a use of the cyclic heptapeptide according to the first aspect of the present invention for preventing and / or treating diseases related to cartilage damage.

[0019] In another preferred embodiment, the chondrocyte damage-related disease has characteristics selected from the group consisting of:

[0020] (a) ACAN gene or protein expression levels are lower than normal;

[0021] (b) Col2a1 gene or protein expression levels are significantly lower than normal levels;

[0022] (c) The expression level of Adamts5 gene or protein is significantly higher than the normal level;

[0023] (d) The expression level of MMP3 gene or protein was significantly higher than the normal level.

[0024] In a sixth aspect, the present invention provides a use of the cyclic heptapeptide according to the first aspect of the present invention for preventing and / or treating osteoarthritis-related diseases.

[0025] In a seventh aspect, the present invention provides a use of the cyclic heptapeptide according to the first aspect of the present invention for preventing and / or treating diseases related to muscle damage.

[0026] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one.

[0027] Compared with the prior art, the present invention has the following technical effects:

[0028] The cyclic peptide provided by the present invention is synthesized into Cyclo-[Thr-Tyr-Val-Pro-Lys-Ala-Phe] by head-to-tail cyclization of a linear peptide. Compared with the linear peptide, the biological activity such as improving cartilage and muscle function is enhanced. The cyclic peptide of the present invention can repair cartilage and underlying bone damage and relieve synovial inflammation, providing a new strategy for the treatment of osteoarthritis; the cyclic peptide of the present invention can repair muscle damage. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Shown is the molecular structural formula of the cyclic peptide Cyclo-[Thr-Tyr-Val-Pro-Lys-Ala-Phe] of the present invention.

[0030] Figure 2 Shown is a graph showing the results of measuring the purity and molecular weight of the cyclic peptide Cyclo-[Thr-Tyr-Val-Pro-Lys-Ala-Phe] of the present invention.

[0031] Figure 3 The results show the evaluation results of cartilage functional indicators by real-time fluorescence quantitative PCR detection of cyclic peptides and linear peptides in the examples of the present invention. Among them, P < 0.05 indicates that the data between the two groups are statistically significant, that is, there is a significant difference.

[0032] Figure 4 The results show the effects of linear peptide (DP) and cyclic peptide (Circle) on muscle function in a mouse grip strength test, as described in an example of the present invention. The values ​​above the lines represent P values, with P < 0.05 indicating statistical significance between the two groups. DETAILED DESCRIPTION

[0033] To make the purpose, technical solutions, beneficial effects, and significant improvements of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, all the embodiments described are only some embodiments of the present invention, not all embodiments; based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0034] the term

[0035] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0036] As used herein, the term “including” or “comprising” encompasses “comprising,” “consisting mainly of,” “consisting essentially of,” and “consisting of;” “consisting mainly of,” “consisting essentially of,” and “consisting of” are subordinate concepts of “containing,” “having,” or “including.”

[0037] As used herein, the terms "linear peptide Thr-Tyr-Val-Pro-Lys-Ala-Phe," "linear peptide," and "linear peptide TYVPKAF" are used interchangeably.

[0038] As used herein, the terms "cyclic peptide Cyclo-[Thr-Tyr-Val-Pro-Lys-Ala-Phe]", "cyclic heptapeptide", and "cyclic peptide Cyclo-[TYVPKAF]" can be used interchangeably.

[0039] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally carried out under conventional conditions or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight. The experimental materials and reagents involved in the present invention can be obtained from commercial sources unless otherwise specified.

[0040] Example 1: Synthesis of Cyclic Heptapeptide Cyclo-[Thr-Tyr-Val-Pro-Lys-Ala-Phe]

[0041] The cyclic peptide Cyclo-[Thr-Tyr-Val-Pro-Lys-Ala-Phe] of the present invention is formed by cyclizing the linear peptide TYVPKAF (SEQ ID NO: 1) by connecting the end to the end. The specific synthesis steps are as follows:

[0042] 1. Solid-phase synthesis stage: Select the resin carrier 2-chlorotrityl chloride resin (2-CTC RESIN), and form an ester bond between the chlorine atom and the carboxyl group of phenylalanine (Phe) to anchor the amino acid to the resin. After each coupling reaction, the side chain protecting group Fmoc is added to protect the α-amino group of Phe with Fmoc to obtain Fmoc-Phe-2-ctc-Resin.

[0043] 2. Remove Fmoc, use piperidine / DMF solution to remove the Fmoc group, expose the α-amino group (-NH2) of Phe, and obtain the resin complex NH2-Phe-2-ctc-Resin with free amino group.

[0044] 3. Connect Fmoc-Ala-OH and activate the carboxyl group of the Fmoc-protected alanine (Fmoc-Ala-OH) (e.g., using HBTU / HOBt) to condense it with the exposed Phe amino group on the resin to form a peptide bond (Ala-Phe) to obtain Fmoc-Ala-Phe-2-ctcResin.

[0045] 4. Repeat steps 2-3 and connect the protected amino acids in sequence to finally obtain NH2-Thr(TBU)-Tyr(TBU)-Val-Pro-Lys(Boc)-Ala-Phe-2-CTC Resin.

[0046] 5. Cleavage of resin 2-CTC RESIN yields NH2-Thr(TBU)-Tyr(TBU)-Val-Pro-Lys(Boc)-Ala-Phe-COOH.

[0047] 6. The amino group and carboxyl group undergo dehydration condensation to form the ring Cyclo-[Thr(TBU)-Tyr(TBU)-Val-Pro-Lys(Boc)-Ala-Phe].

[0048] 7. Remove the protecting group to obtain the crude cyclic peptide Cyclo-[Thr-Tyr-Val-Pro-Lys-Ala-Phe].

[0049] 8. Purification by high performance liquid chromatography (HPLC) and freeze-drying gave the fine cyclic peptide Cyclo-[Thr-Tyr-Val-Pro-Lys-Ala-Phe] with a purity of 96.5984%. Mass spectrometry (MS) determined the molecular weight of Cyclo-[Thr-Tyr-Val-Pro-Lys-Ala-Phe] to be 807.80 Da ( Figure 2 ).

[0050] Example 2: Effects of the Cyclic Heptapeptide of the Present Invention on Primary Chondrocytes

[0051] 1. Incubate mouse primary chondrocytes to 80%-90% of the area of ​​a 6-well cell culture plate (approximately 1x10 6 ~2x10 6 cells), and then divided them into 4 groups, as follows:

[0052] (1) Ctrl group: healthy primary chondrocytes;

[0053] (2) IL-1β (10 ng / mL) group: 10 ng / mL IL-1β was added and incubated for 2 hours to establish a primary chondrocyte inflammatory injury model;

[0054] (3) 1 mM linear peptide TYVPKAF group (DP (1 mM)): 10 ng / mL IL-1β was added and incubated with 20 mM linear peptide TYVPKAF for 2 h;

[0055] (4) 20 mM linear peptide TYVPKAF group (DP (20 mM)): 10 ng / mL IL-1β was added and incubated with 20 mM linear peptide TYVPKAF for 2 hours;

[0056] (5) 1 mM cyclic peptide Cyclo-[Thr-Tyr-Val-Pro-Lys-Ala-Phe] group (Circle (1 mM)): 10 ng / mL IL-1β was added and incubated with 1 mM Cyclo-[Thr-Tyr-Val-Pro-Lys-Ala-Phe] for 2 hours).

[0057] After reverse transcription of RNA extracted from each component in the 6-well plate, real-time fluorescence quantitative PCR was performed to measure key indicators of cartilage function (ACAN, Col2a1, Adamts5, MMP3).

[0058] The results showed that 20 mM linear peptide TYVPKAF and 1 mM cyclic peptide Cyclo-[Thr-Tyr-Val-Pro-Lys-Ala-Phe] significantly upregulated the expression levels of ACAN and Col2a1 and reduced the transcription levels of Adamts5 and MMP3, while 1 mM linear peptide TYVPKAF had no such effects. This indicates that high concentrations of linear peptide TYVPKAF and low concentrations of cyclic peptide Cyclo-[Thr-Tyr-Val-Pro-Lys-Ala-Phe] significantly improved cartilage function, and under the same low concentration conditions (both 1 mM), compared with the linear peptide TYVPKAF, the cyclic peptide Cyclo-[Thr-Tyr-Val-Pro-Lys-Ala-Phe] had stronger functional activity ( Figure 3 ).

[0059] Example 3: Effect of the Cyclic Heptapeptide of the Present Invention on Muscle Damage in Mice

[0060] The experimental groups of this embodiment are as follows:

[0061] (1) C57BL / 6J mice were injected with 125 μL of normal saline, which was the Vehicle group;

[0062] (2) Muscle injury model was induced by intramuscular injection of 125 μL of 1.2% BaCl2 solution into C57BL / 6J mice, namely the BaCl2 group.

[0063] (3) C57BL / 6J mice were injected intramuscularly with 125 μL of 1.2% BaCl2 solution one day later, and then 125 μL of 20 mM linear peptide TYVPKAF (with saline as solvent) was injected intramuscularly to form the DP group.

[0064] (4) C57BL / 6J mice were injected intramuscularly with 125 μL of 1.2% BaCl2 solution one day later, and then 125 μL of 1 mM cyclic peptide Cyclo-[Thr-Tyr-Val-Pro-Lys-Ala-Phe] (with saline as solvent) was injected intramuscularly to form the Circle group.

[0065] After one week of feeding, muscle damage was detected by grip strength test.

[0066] The results showed that 20 mM linear peptide TYVPKAF had no effect on muscle damage; 1 mM cyclic peptide Cyclo-[Thr-Tyr-Val-Pro-Lys-Ala-Phe] had a significant improvement effect on muscle damage ( Figure 4 ).

[0067] The above describes the preferred embodiments of the present invention. It should be understood that the present invention is not limited to the above-mentioned specific embodiments, and the devices and structures that are not described in detail should be understood to be implemented in a common manner in the art; any technician familiar with the art can use the above-mentioned disclosed methods and technical contents to make many possible changes and modifications to the technical solutions of the present invention without departing from the scope of the technical solutions of the present invention, or modify them into equivalent embodiments of equivalent changes, which does not affect the essential content of the present invention. Therefore, any simple modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention that do not depart from the content of the technical solutions of the present invention are still within the scope of protection of the technical solutions of the present invention.

Claims

1. A cyclic peptide, characterized in that The cyclic peptide is a cyclic heptapeptide consisting of an amino acid sequence as shown in SEQ ID NO.1: Thr-Tyr-Val-Pro-Lys-Ala-Phe, and the structural formula of the cyclic heptapeptide is: 。 2. The cyclic peptide according to claim 1, characterized in that The cyclization mode of the cyclic peptide is that the threonine at the 1st position and the phenylalanine at the 7th position of the linear peptide form a peptide bond through dehydration condensation, thereby forming a cyclic structure.

3. A gene sequence encoding the cyclic peptide according to claim 1. A fusion protein comprising the cyclic peptide according to claim 1.

5. A method for preparing the cyclic peptide according to claim 1, characterized in that: The method involves solid phase synthesis.

6. The method according to claim 5, characterized in that The method comprises the following steps: (1) Solid-phase synthesis stage: Select the resin carrier 2-chlorotrityl chloride resin (2-CTC RESIN), and form an ester bond between the chlorine atom and the carboxyl group of phenylalanine (Phe) to anchor the amino acid on the resin; and add the side chain protecting group Fmoc after each coupling reaction to protect the amino group of Phe with Fmoc to obtain the synthetic compound Fmoc-Phe-2-ctc-Resin; (2) Fmoc removal: Use piperidine / DMF solution to remove the Fmoc group, exposing the amino group of Phe to obtain the resin complex NH2-Phe-2-ctc-Resin with free amino group; (3) Connecting Fmoc-Ala-OH: Activate the carboxyl group of Fmoc-protected alanine (Fmoc-Ala-OH) and condense it with the amino group of the Phe exposed on the resin in step (2) to form a peptide bond to obtain Fmoc-Ala-Phe-2-ctc Resin; (4) After sequentially connecting the protected amino acids, the resin is cleaved and the amino and carboxyl groups are dehydrated and condensed to obtain the cyclic peptide according to claim 1.

7. The use of the cyclic peptide according to claim 1, characterized in that Used for preventing and / or treating diseases related to cartilage damage.

8. The use according to claim 7, characterized in that The cartilage damage-related disease has a characteristic selected from the group consisting of: (a) ACAN gene or protein expression levels are lower than normal; (b) Col2a1 gene or protein expression levels are significantly lower than normal levels; (c) The expression level of Adamts5 gene or protein is significantly higher than the normal level; (d) The expression level of MMP3 gene or protein was significantly higher than the normal level.

9. The use of the cyclic peptide according to claim 1, characterized in that For the prevention and / or treatment of osteoarthritis-related diseases.

10. Use of the cyclic peptide according to claim 1, characterized in that For the prevention and / or treatment of muscle damage-related diseases.

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