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Stapled peptides for restraining osteoclast differentiation and preparation method and application of stapled peptides for restraining osteoclast differentiation

A technology for inhibiting osteoclasts and stapling peptides, applied in the field of polypeptide drugs, can solve problems such as the role of osteoclast differentiation not seen, and achieve the effect of high yield

Active Publication Date: 2020-06-05
SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no research on the effect of FRATtide or its staple peptide on osteoclast differentiation

Method used

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  • Stapled peptides for restraining osteoclast differentiation and preparation method and application of stapled peptides for restraining osteoclast differentiation
  • Stapled peptides for restraining osteoclast differentiation and preparation method and application of stapled peptides for restraining osteoclast differentiation
  • Stapled peptides for restraining osteoclast differentiation and preparation method and application of stapled peptides for restraining osteoclast differentiation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] Example 1 Preparation of Staple Peptide Inhibiting Osteoclast Differentiation in the Present Invention

[0067] 1. Synthesis of stapled peptide

[0068] Such as figure 2 Shown:

[0069] (1) Preparation of compound 1

[0070] Take 500 mg of amino resin (sample loading is 0.30 mmol g -1 ) into the solid-phase synthesis reaction tube, soaked in DCM for 20 minutes to fully swell the resin, and drained for later use.

[0071] Add 20% piperidine-DMF solution (0.1M Oxyme) until the resin is completely submerged, shake at 25°C for 5 min×2 to remove Fmoc on the resin, and wash the resin with DCM and DMF for 3 times.

[0072] (2) Preparation of Compound 2

[0073] The first amino acid in the sequence (1mmol), Oxyme (142mg, 1mmol) and DIC (155.0μL, 1mmol) were mixed in 6ml NMP, added to the resin and shaken at 60°C for 20min (S 5 The last amino acid was reacted for 1 h, and the reaction was repeated once), and the resin was washed with DCM and DMF successively for 3 times e...

Embodiment 2

[0086] Identification and structure analysis of embodiment 2 product

[0087] The product obtained in step 2 of Example 1 was identified by HPLC and structurally analyzed by HR-Q-TOF-MS (high-resolution matrix-assisted laser desorption ionization time-of-flight mass spectrometry), and the chromatographic mobile phase was acetonitrile and water. Mobile phase A is an aqueous solution with a volume fraction of 0.1% TFA, and mobile phase B is an acetonitrile solution with a volume fraction of 0.1% TFA, gradient elution (0-5min, mobile phase B: 5%; 5-30min, mobile phase B: 5%~90%); flow rate 15.0mL·min -1 ; The detection wavelengths are 214nm and 254nm, and the injection volume is 20μl. It is determined that it is consistent with the peak time of the main peak of the crude product, and the purity of the staple peptide prepared by this method is >98% ( Figure 3-4 ). The results were analyzed by HR-ESI-MS mass spectrometer as Figure 5-12 shown.

Embodiment 3

[0088] Example 3 Inhibition of osteoclast differentiation experiment

[0089] TRAP staining assay: Osteoclast formation was quantified by TRAP-positive cells. BMMs were seeded into 96-well plates at a density of 8000 cells per well. After incubation for 24 h, BMMs were incubated with M-CSF at a concentration of 30 ng / ml, RANKL at 50 ng / mL and different concentrations of peptides (0, 20 and 40 μM). Fresh medium was replaced every 2 days. After 5 days of osteoclast induction, wash twice with PBS, fix in 4% paraformaldehyde (pH 7.4) at room temperature for 10 min, and stain with TRAP staining kit. Positive multinucleated osteoclasts were counted under a light microscope, and the percentage of osteoclasts in each well was measured using Image J software.

[0090] see results Figure 13-14 , the staple peptide of the present invention can inhibit the differentiation of osteoclasts, among which FRN-2, FRC-1, FRC-2 and FRC-3 have the most prominent effects, and FRC-2 has the best...

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Abstract

The invention relates to stapled peptides for restraining osteoclast differentiation and a preparation method and application of the stapled peptides for restraining osteoclast differentiation. Aminoresins are used as a carrier, according to the amino acid sequence of a formwork FRATtide:Ac-DPHRLLQQLVLSGNLIKEAVRRLHSR-NH2 in a DIC-Oxime condensation system, through an Fmoc solid phase synthesis method, a peptide chain is obtained by synthetizing, based on reserving key amino acid residues, S5 is used for replacing original amino acid in the specific positions, and liner-chain peptides connected to the resin are subjected to olefin metathesis reaction and cyclization in a dichloroethane solution of a Grubbs I reagent, and then are cut down from the resin, so that the target stapled peptidesare obtained. The method disclosed by the invention is simple to realize, and high in production rate, and the stapled peptides are high in purity. Further experiment confirms that the stapled peptides can notably restrain osteoclast differentiation, and have potential application value in treatment of relevant diseases of osteoporosis and the like.

Description

technical field [0001] The invention belongs to the field of polypeptide drugs, and in particular relates to a staple peptide for inhibiting osteoclast differentiation and its preparation method and application. Background technique [0002] Osteoporosis is a bone disease characterized by decreased bone mass, changes in the microstructure of bone tissue, increased bone fragility, decreased bone strength, and susceptibility to fracture. Its pathophysiological basis is the imbalance between bone resorption and bone formation. This balance is regulated by osteoblasts, which promote bone formation, and osteoclasts, which promote bone resorption. Currently, the most widely used anti-osteoporosis drugs include small molecule drugs such as bisphosphonates that inhibit osteoclast differentiation and teriparatide that promote bone formation. In order to improve the selectivity of therapeutic drugs and reduce side effects, regulating the important signaling pathways related to osteob...

Claims

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Application Information

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IPC IPC(8): C07K7/06C07K7/08C07K1/06C07K1/04C12N5/078A61K38/08A61P19/10
CPCC07K7/06C07K7/08C12N5/0643A61P19/10A61K38/00Y02P20/55
Inventor 李翔廖洪利刘泰容陈思丛薇廖秀飞胡宏岗张卫东
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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