G5-like peptide for promoting bone formation or medicinal salt thereof

Abstract

The invention belongs to the field of polypeptide drugs, and particularly relates to a G5-like peptide for promoting bone formation or a pharmaceutically acceptable salt thereof. The similar peptide or the medicinal salt thereof comprises the following amino acid sequence or consists of the following amino acid sequence: 1) G5-1: SEQ ID NO: 1; the invention relates to a G5-like peptide or a pharmaceutically acceptable salt thereof, in particular to a G5-like peptide or a pharmaceutically acceptable salt thereof, the G5-like peptide or the pharmaceutically acceptable salt thereof has the advantages that the G5-like peptide or the pharmaceutically acceptable salt thereof can obviously up-regulate expression of alkaline phosphatase and obviously promote osteogenic differentiation capacity, has potential clinical applicability and research value in osteoporosis treatment, and is non-toxic to other tissues, safe and reliable.

Description

Technical Field

[0001] The invention belongs to the field of polypeptide drugs, and specifically relates to a G5-like peptide or a pharmaceutically acceptable salt thereof for promoting bone formation. Background Art

[0002] Bone regeneration is a complex process involving a combination of many biological factors. Growth factors have been implicated in bone repair / regeneration and are biologically active peptide or protein hormones that influence immune function as well as proliferation, chemotaxis, and differentiation of epithelial cells, osteocytes, and connective tissue cells. These hormones bind to specific cell surface tyrosine kinase receptors that are present on a variety of target cells, including osteoblasts, osteoblasts, and fibroblasts.

[0003] Among the growth factors related to bone repair / regeneration, an important growth factor called osteogenic growth peptide (OGP) has been widely studied. OGP is a polypeptide composed of 14 amino acid residues. It is a promoter of systemic response to bone marrow damage and exists in mammalian serum at micromolar concentrations. Studies have found that its C-terminal OGP (10-14) pentapeptide (hereinafter referred to as G5 peptide) is the minimum amino acid sequence that maintains all OGP-like activities. Literature shows that OGP / G5 plays an important role in bone repair / regeneration, mainly in stimulating the proliferation, differentiation, alkaline phosphatase activity and matrix mineralization of osteoblastic cell lineage cells. In addition, OGP regulates the expression of transforming growth factor (TGF), insulin-like growth factor (IGF) and basic fibroblast growth factor (bFGF), thereby increasing bone formation and trabecular bone density in vivo. OGP can stimulate the activity of osteoblasts and promote the growth of osteoblasts and fibroblasts. G5 is of great value in the research and treatment of osteoporosis.

[0004] The use of small molecule peptides for disease treatment has become a new trend. This study developed G5-like peptides for osteoporosis research and osteogenesis, providing new drug options for osteoporosis treatment. Summary of the invention

[0005] The present invention provides a G5-like peptide or a pharmaceutically acceptable salt thereof for promoting bone formation, wherein the G5-like peptide or a pharmaceutically acceptable salt thereof comprises or consists of the amino acid sequence shown below: 1) SEQ ID NO: 1; 2) SEQ ID NO: 2; 3) SEQ ID NO: 3; or 4) SEQ ID NO: 4.

[0006] Preferably, the G5-like peptide is further coupled to a functional domain that promotes bone formation.

[0007] Preferably, the amino acid sequence of the bone formation promoting domain is as shown in SEQ ID NO:5.

[0008] Preferably, the G5-like peptide is directly coupled to the bone formation promoting functional domain via a peptide bond.

[0009] Preferably, the G5 analog peptide or a pharmaceutically acceptable salt thereof comprises or consists of the amino acid sequence shown below: G5-1: SEQ ID NO: 6; G5-2: SEQ ID NO: 7; G5-3: SEQ ID NO: 8; or G5-4: SEQ ID NO:9.

[0010] In a preferred embodiment of the present invention, the present invention provides a pharmaceutical composition, which comprises the bifunctional polypeptide of the present invention or a pharmaceutically acceptable salt thereof.

[0011] Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient.

[0012] Preferably, the pharmaceutical composition is used to promote osteogenic differentiation or treat osteoporosis.

[0013] In another preferred embodiment of the present invention, the present invention provides the use of the bifunctional polypeptide of the present invention or its pharmaceutically acceptable salt and pharmaceutical composition thereof in the preparation of drugs for treating osteoporosis and / or promoting osteogenic differentiation.

[0014] Preferably, the treatment of osteoporosis is achieved by promoting bone differentiation.

[0015] The present invention develops a G5-like peptide and directly fuses it with SESSE that promotes bone formation to obtain a fusion polypeptide, which significantly upregulates the expression of alkaline phosphatase and significantly promotes the ability of osteogenic differentiation. It has potential clinical applicability and research value in the treatment of osteoporosis, is non-toxic to other tissues, and is safe and reliable.

[0016] The concept, specific structure and technical effects of the present invention will be further described below in conjunction with the accompanying drawings to fully understand the purpose, characteristics and effects of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is the result of CCK-8 testing of the toxicity of G5-X (X=1 / 2 / 3 / 4).

[0018] Figure 2These are the results of ALP staining of hBMSC cells induced by compound G5-X (X=1 / 2 / 3 / 4) after 14 days of induction, where A: no induction solution added; B: induction solution added; C: induction solution + SESSE; D: induction solution + G5-1; E: induction solution + G5-2; F: induction solution + G5-3; G: induction solution + G5-4.

[0019] Figure 3 This is the expression of osteogenic-related genes in hBMSC cells induced by compound G5-X (X=1 / 2 / 3 / 4) for 14 days. DETAILED DESCRIPTION

[0020] The present invention is described in detail below in conjunction with the accompanying drawings and specific examples, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0021] Example 1 Synthesis of agonist peptides

[0022] 1. Experimental Materials The materials and reagents used in the present invention are purchased from commercial products. Taking G5-1 as an example, the synthesis preparation method of the agonist peptide in the present invention is described (the preparation of other agonist peptides in the present invention only requires replacing the synthesis sequence of amino acid raw materials).

[0023] 2. Weigh 15 mmol equivalent of resin and put it into the reactor. Add DCM (dichloromethane) to swell for half an hour, then remove DCM, add the first amino acid in the sequence (15 mmol), DIEA (diisopropylethylamine), appropriate amount of DMF (dimethylformamide), DCM solution (appropriate amount means that the resin can be fully agitated), and react with nitrogen bubbling for 60 minutes. Then add about 75 mmol equivalent of methanol, react for half an hour, remove the reaction solution, and wash with DMF and DCM.

[0024] 3. Add appropriate amount of 20% piperidine / DMF to remove Fmoc (9-fluorenylmethoxycarbonyl) protecting group, wash, and detect with detection reagent.

[0025] 4. Add the second amino acid in the sequence (also 45 mmol), 45 mmol HBTU (benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate) and DIEA into the reactor, bubble nitrogen for half an hour, draw out the liquid, wash with DMF and DCM, and detect with ninhydrin.

[0026] 5. Add the amino acids in the sequence in the same way as in steps 3 and 4, remove the liquid, wash with DMF, and test with the detection reagent.

[0027] 6. After blowing the resin dry with nitrogen, remove it from the reaction column, weigh it, pour it into a flask, and then add a certain amount of 95% TFA (trifluoroacetic acid) cutting solution into the flask and shake it for 2 hours. The purpose is to cleave the polypeptide from the resin carrier and remove the side chain protecting groups of the amino acids.

[0028] 7. Filter off the resin to obtain a filtrate, then add a large amount of ether to the filtrate to precipitate a crude product, then centrifuge and wash to obtain the crude product of the sequence.

[0029] 8. Analysis, purification and mass spectrometry: Use ESI (electrospray ionization) ion source mass spectrometer to detect the correctness of the molecular weight of the sequence, and use high performance liquid chromatography to purify the crude product to the required purity.

[0030] 9. Collect the purified target polypeptide solution and put it into a freeze dryer for concentration, and freeze-dry it into white powder.

[0031] 10. Preparation results After the preparation was completed, the molecular weight test results of each agonist peptide compound were shown in Table 1, and the results showed that the prepared compounds were all correct.

[0032] Table 1 List of synthetic agonist peptide compounds and molecular weight

[0033] Example 2 Toxicity test of peptide compounds

[0034] 1. Experimental Materials The materials and reagents used in this evaluation were purchased from commercial products.

[0035] 2. Experimental methods and results Taking compound G5-X (X=1 / 2 / 3 / 4) as an example, the toxicity test of peptide compounds is carried out as follows: hBMSCs in the logarithmic growth phase were plated at 5000 cells / mL in a 96-well plate, and peptide compounds were added to the 96-well plate at different concentrations, with 3 replicates for each concentration. After incubation in a carbon dioxide incubator for 24 hours, the culture medium was discarded, CCK-8 reagent was added, and detection was performed at a wavelength of 450nm. The results are shown in Figure 1 As shown, there was no statistical difference between the different concentration groups compared with the group without peptide addition, which indicates that the addition of G5-like peptides has no toxicity to cells.

[0036] Example 3 Osteogenesis induction of hBMSC cells by peptide compounds and ALP staining

[0037] 1. Experimental Materials The materials and reagents used in this evaluation were purchased from commercial products.

[0038] 2. Experimental methods and results Taking compound G5-X (X=1 / 2 / 3 / 4) as an example, the osteoclast induction test is performed as follows: hBMSC cells were used for osteogenic induction. The cells were seeded in 12-well plates and divided into a normal culture group (without induction solution), an induction group (with induction solution), and an experimental group (with induction solution plus peptides). The induction solution was DMEM / F12 medium containing β-glycerophosphate sodium and vitamin C. After 14 days of induction, ALP staining was performed. The results are shown in Figure 2 As shown, compared with the induced group, the alkaline phosphatase content in the experimental group was increased, which indicated that the addition of G5-X (X=1 / 2 / 3 / 4) promoted the ability of osteogenic differentiation.

[0039] Example 4 Effect of peptide compounds on osteogenesis-related gene expression

[0040] 1. Experimental Materials The materials and reagents used in this evaluation were purchased from commercial products.

[0041] 2. Experimental methods and results Taking compound G5-X (X=1 / 2 / 3 / 4) as an example, osteoclast gene expression test is performed as follows: hBMSC cells were used for osteogenic induction. The cells were seeded in 12-well plates and divided into a normal culture group (without induction solution), an induction group (with induction solution), and an experimental group (with induction solution plus peptides). The induction solution was DMEM / F12 medium containing sodium β-glycerophosphate and vitamin C. After 14 days of induction, the total RNA of the cells was collected for qPCR. The results are shown in Figure 3 As shown, the expression of ALPL in the experimental groups was significantly increased compared with that in the induced group, indicating that the addition of G5-X (X=1 / 2 / 3 / 4) promoted the ability of osteogenic differentiation.

[0042] The preferred specific embodiments of the present invention are described in detail above. It should be understood that ordinary technicians in the field can make many modifications and changes based on the concept of the present invention without creative work. Therefore, all technical solutions that can be obtained by technicians in the technical field based on the concept of the present invention through logical analysis, reasoning or limited experiments on the basis of the prior art should be within the scope of protection determined by the claims.

Claims

1. A G5-like peptide or a pharmaceutically acceptable salt thereof for promoting bone formation, wherein the G5-like peptide or a pharmaceutically acceptable salt thereof comprises or consists of the amino acid sequence shown below: 1) SEQ ID NO: 1; 2) SEQ ID NO: 2; 3) SEQ ID NO: 3; or 4) SEQ ID NO:

4.

2. The G5-like peptide or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: The G5-like peptide is also coupled to a functional domain that promotes bone formation.

3. The G5-like peptide or a pharmaceutically acceptable salt thereof according to claim 2, characterized in that: The amino acid sequence of the bone formation promoting functional domain is shown in SEQ ID NO:

5.

4. The G5-like peptide or a pharmaceutically acceptable salt thereof according to claim 3, characterized in that: The G5-like peptide is directly coupled to the bone formation promoting functional domain via a peptide bond.

5. The G5-like peptide or a pharmaceutically acceptable salt thereof according to claim 4, characterized in that: The G5-like peptide or a pharmaceutically acceptable salt thereof comprises or consists of the amino acid sequence shown below: G5-1: SEQ ID NO: 6; G5-2: SEQ ID NO: 7; G5-3: SEQ ID NO: 8; or G5-4: SEQ ID NO:

9.

6. A pharmaceutical composition comprising the G5 analog peptide or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 5.

7. The pharmaceutical composition according to claim 6, characterized in that The pharmaceutical composition further includes a pharmaceutically acceptable carrier or excipient.

8. The pharmaceutical composition according to claim 6, characterized in that The pharmaceutical composition is used for promoting osteogenic differentiation or treating osteoporosis.

9. Use of the G5 analog peptide or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 5 and / or the pharmaceutical composition according to any one of claims 6 to 8 in the preparation of a drug for treating osteoporosis and / or promoting osteogenic differentiation.

10. The use according to claim 9, characterized in that: The treatment of osteoporosis is achieved by promoting bone differentiation.

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