Small molecule peptide and application thereof

Abstract

The invention discloses a small molecule peptide and an application thereof. The small molecule peptide is TYVPKAF. The small molecule peptide TYVPKAF is screened from pilose antler for the first time, and the small molecule peptide TYVPKAF is suitable for oral administration, high in bioavailability, high in in-vivo efficacy, strong in selectivity and free of toxic and side effects. The small molecule peptide TYVPKAF provided by the invention has the advantages that the expression levels of MMP13 and cGAS are inhibited in a targeted manner, cartilage and lower bone injury are repaired, synovitis is relieved, and a new strategy is provided for treatment of osteoarthritis (OA). According to the small molecule peptide TYVPKAF, bone formation is promoted by inhibiting the Fap expression level, so that the healing process of bone defects is accelerated, and a new strategy is provided for treatment of the bone defects.

Description

Technical Field

[0001] The present invention relates to the technical field of bioactive peptides, and in particular to a small molecule peptide and application thereof. Background Art

[0002] Osteoarthritis (OA) is a bone degenerative disease characterized by cartilage and underlying bone damage and synovial inflammation. There are no anti-osteoarthritis drugs (DMOADs) approved by the National Medical Products Administration (NMPA). Currently, clinical medications only relieve symptoms but do not block the overall course of OA. Most patients rely on artificial joint replacement surgery to restore joint function, which places a heavy burden on patients, their families, and the social health system.

[0003] Globally, approximately 10% to 20% of patients with bone defects cannot recover. How to heal bone defects caused by bone degenerative diseases, severe trauma, infection and surgery has become a difficult challenge for clinical surgeons. Osteogenic differentiation is a key step in bone formation, which is a complex process of osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) and then differentiation of osteoblasts into osteocytes. In addition, inhibiting bone resorption is also an important part of bone defect repair.

[0004] Natural active peptides and their derivatives with functional properties have attracted much attention in drug development due to their advantages such as short half-life, low toxicity and side effects, and low immunogenicity. Although natural peptides such as insulin and thymopentin have good therapeutic effects, most of them have low bioavailability due to protease hydrolysis, physiological barriers and transcellular transport barriers. Without the help of special excipients or nanocarriers, they can only be administered by injection. Gastrointestinal administration (oral, rectal, etc.) enhances patients' compliance with medication.

[0005] Deer antler has strong tissue regeneration and repair capabilities. Therefore, if active small molecule peptides suitable for oral administration and capable of treating osteoarthritis and bone defects are screened out from deer antler, it can provide an important basis for the development of oral drugs for the treatment of osteoarthritis and bone defects. Summary of the invention

[0006] The purpose of the present invention is to provide a small molecule peptide and its application in view of the deficiencies in the prior art.

[0007] To achieve the above object, the technical solution adopted by the present invention is:

[0008] The first aspect is to provide a small molecule peptide, the amino acid sequence of which is TYVPKAF as shown in SEQ ID NO: 1.

[0009] The second aspect is to provide the use of the above-mentioned small molecule peptide in the preparation of drugs for treating osteoarthritis.

[0010] The third aspect is to provide a drug for treating osteoarthritis, which uses the above-mentioned small molecule peptide as the only active ingredient.

[0011] Furthermore, the dosage form of the drug is an oral dosage form.

[0012] The fourth aspect is to provide the use of the above-mentioned small molecule peptide in the preparation of drugs for treating bone defects.

[0013] The fifth aspect is to provide a drug for treating bone defects, with the above-mentioned small molecule peptide as the only active ingredient.

[0014] Furthermore, the dosage form of the drug is an oral dosage form.

[0015] The present invention adopts the above technical solution, and has the following technical effects compared with the prior art:

[0016] The present invention screens out a small molecule peptide TYVPKAF from deer antler for the first time. The small molecule peptide TYVPKAF is suitable for oral administration and has high bioavailability, high in vivo efficacy, strong selectivity and no toxic side effects.

[0017] The small molecule peptide TYVPKAF of the present invention targets and inhibits the expression levels of MMP13 and cGAS, repairs cartilage and underlying bone damage and relieves synovial inflammation, and provides a new strategy for the treatment of osteoarthritis (OA).

[0018] The small molecule peptide TYVPKAF of the present invention promotes bone formation by inhibiting the expression level of Fap, thereby accelerating the healing process of bone defects and providing a new strategy for the treatment of bone defects. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 It is the chemical structural formula of the small molecule peptide TYVPKAF.

[0020] Figure 2 The toxic effect of the small molecule peptide TYVPKAF in Example 2 on primary chondrocytes.

[0021] Figure 3 This is the effect of the small molecule peptide TYVPKAF in Example 2 on the expression level of MMP13 gene transcription.

[0022] Figure 4 This is the effect of the small molecule peptide TYVPKAF in Example 2 on the expression levels of MMP13 and cGAS proteins.

[0023] Figure 5 This is the effect of oral administration of the small molecule peptide TYVPKAF in Example 2 to OA mice.

[0024] Figure 6 The toxic and side effects of the small molecule peptide TYVPKAF in Example 3 on MC-3T3E1.

[0025] Figure 7 This is the ALP staining result of MC-3T3E1 after using the small molecule peptide TYVPKAF in Example 3.

[0026] Figure 8 This is the inhibitory effect of the small molecule peptide TYVPKAF in Example 3 on Fap activity.

[0027] Fig. 9 This is the effect of oral administration of the small molecule peptide TYVPKAF in Example 3 to mice with bone defects. DETAILED DESCRIPTION

[0028] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments, but is not intended to be limiting of the present invention. It should be noted that the embodiments and features in the embodiments of the present invention may be combined with each other without conflict.

[0029] Example 1

[0030] 1. Matrix metalloproteinase-13 (MMP-13) is overexpressed in chondrocytes and synovial cells, and preferentially destroys the homeostasis of cartilage and its internal environment by cleaving type II collagen, which is considered to be a key factor leading to human OA. Inhibition of MMP13 can repair cartilage damage and its internal environment, thereby regulating the progression of OA.

[0031] In addition, the cyclic GMP-AMPsynthase-stimulator ofinterferon gene (cGAS-STING) signaling pathway, as an important component of the innate immune system, is the main pathway for cytoplasmic DNA recognition. cGAS can be triggered by a variety of cytoplasmic double-stranded DNA, becoming an important bridge connecting autoimmunity, inflammatory response and cell apoptosis. Inhibiting the cGAS-STING pathway or its downstream signaling pathways can reduce synovial inflammation, thereby improving OA symptoms.

[0032] Based on in vivo and in vitro digestion and metabolism models, the present invention screened and identified a variety of suitable oral small molecule peptides from velvet antler source proteins by high performance liquid chromatography coupled with a high-resolution mass spectrometer and peptide identification software, predicted the inhibitory potential of small molecule peptides on MMP13 and cGAS through molecular docking, and screened out the best active small molecule peptide TYVPKAF (Thr-Tyr-Val-Pro-Lys-Ala-Glu).

[0033] 2. Osteogenesis inhibitory factor is fibroblast activation protein-α (Fap). Inhibiting its activity can promote bone formation and inhibit bone resorption. It has a synergistic effect with PTH1-34 and is a new potential drug target for repairing bone defects.

[0034] Based on in vivo and in vitro digestion and metabolism models, the present invention screened and identified a variety of small molecule peptides suitable for oral administration from velvet antler protein, predicted the inhibitory potential of small molecule peptides on Fap through molecular docking, and established the optimal small molecule peptide TYVPKAF (Thr-Tyr-Val-Pro-Lys-Ala-Glu).

[0035] Example 2

[0036] 1 g of the small molecule peptide TYVPKAF was synthesized in solid phase and diluted to 50 mM, 10 mM, 5 mM, and 1 mM with primary cell culture medium. The small molecule peptide TYVPKAF was added to the primary chondrocyte culture dish, while the control group without the small molecule peptide TYVPKAF was used as the normal control group. The safety concentration of the small molecule peptide was evaluated based on primary chondrocytes. The culture dishes were incubated in an incubator (37°C, 5% CO 2 After incubation for 1 to 2 days, the cell viability test kit (CCK-8) was used to analyze the viability of primary chondrocytes to determine the toxicity of the small molecule peptide TYVPKAF to chondrocytes. The results showed that ( Figure 2 ), compared with the normal control group, within the concentration range of 0-50mM, the small molecule peptide TYVPKAF had no toxic side effects on primary chondrocytes.

[0037] Figure 3 and Figure 4 The results showed that based on the OA model of primary chondrocytes induced by IL-1β (10ng / mL), the small molecule peptide TYVPKAF significantly inhibited the gene transcription level of MMP13 in a concentration-dependent manner at a concentration of 1-10mM, and 10mM small molecule peptide TYVPKAF significantly inhibited the expression levels of MMP13 and cGAS proteins. Based on the in vivo and in vitro OA model, real-time fluorescence quantitative PCR confirmed that 10mM small molecule peptide TYVPKAF significantly inhibited the gene transcription level of MMP13; protein immunoblotting confirmed that 10mM small molecule peptide TYVPKAF significantly inhibited the protein expression levels of MMP13 and cGAS.

[0038] The drug was diluted with ordinary drinking water at a concentration of 1 mg / kg and orally administered to mice with post-traumatic osteoarthritis induced by anterior cruciate ligament resection (ACLT) for one month (oral administration group), and compared with the blank control group (Sham group) and the solvent control group (Vehicle group). Safranin O-fast green and H&E staining can intuitively reflect the structure and inflammation of articular cartilage and subchondral bone tissue. The results showed that ( Figure 5 ), compared with the Vehicle group, the oral administration group significantly and effectively promoted cartilage repair and elimination of synovial inflammation, and restored the cartilage and its internal environment of OA mice to a level basically consistent with that of normal mice.

[0039] Example 3

[0040] The small molecule peptide TYVPKAF 1g was synthesized in solid phase and diluted to 50mM, 10mM, 5mM, and 1mM with MC-3T3E1 cell culture medium, and then added to MC-3T3E1 cell culture dishes, respectively. The control group without small molecule peptide TYVPKAF was used as the normal control group. The safety concentration of small molecule peptide was evaluated based on MC-3T3E1 cells. The cells were incubated in an incubator (37°C, 5% CO 2 After incubation for 1 to 2 days, the cell viability of MC-3T3E1 cells was analyzed by a cell viability detection kit (CCK-8) to determine the toxicity of the small molecule peptide TYVPKAF to MC-3T3E1 cells ( Figure 6 ). Diethylnitrosamine (n-nitroso-diethylamin, DEN) and fibroblast activation protein (FAP) inhibitor (Fapi) were added to the culture dish with the culture medium, and the medium was changed every 2 to 3 days. Based on the in vitro osteogenic differentiation model of 7-day embryonic osteoblast precursor cells MC-3T3E1, alkaline phosphatase staining (ALP) found that 10mM concentration had a significant promoting effect on osteogenic differentiation ( Figure 7 ); Western blotting confirmed the inhibitory effect of the small molecule peptide TYVPKAF on Fap ( Figure 8 ).

[0041] The drug was diluted with ordinary drinking water at a concentration of 1 mg / kg and administered orally to mice with non-critical bone defects (0.6 mm in diameter) every day for one month (oral group), and compared with the blank control group (Sham group), the solvent control group (Vehicle group) and the intravenous injection of Fapi twice a week (Fapi injection group). One month later, Micro-CT scanning was used to observe the healing of the bone defect and the repair ability of the small molecule peptide TYVPKAF on the bone defect after oral administration. The results showed that ( Fig. 9 ), the oral administration group promoted the repair of bone defects more effectively than the Fapi injection group.

[0042] The above description is only a preferred embodiment of the present invention, and does not limit the implementation mode and protection scope of the present invention. For those skilled in the art, it should be aware that all solutions obtained by equivalent substitutions and obvious changes made using the contents of the specification and illustrations of the present invention should be included in the protection scope of the present invention.

Claims

1. A small molecule peptide, It is characterized in that The amino acid sequence of the small molecule peptide is shown in SEQ ID NO: 1, which is TYVPKAF.

2. Use of the small molecule peptide as claimed in claim 1 in the preparation of drugs for treating osteoarthritis.

3. A drug for treating osteoarthritis, It is characterized in that The small molecule peptide described in claim 1 is the only active ingredient.

4. The drug for treating osteoarthritis according to claim 3, It is characterized in that The dosage form of the drug is oral dosage.

5. Use of the small molecule peptide as claimed in claim 1 in the preparation of a drug for treating bone defects.

6. A drug for treating bone defects, It is characterized in that The small molecule peptide described in claim 1 is the only active ingredient.

7. The drug for treating bone defects according to claim 6, It is characterized in that The dosage form of the drug is oral dosage.

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