A stapled peptide that inhibits osteoclast differentiation and its preparation method and application

A technology for inhibiting osteoclasts and stapling peptides, applied in the field of polypeptide drugs, can solve the problem of no osteoclast differentiation effect, and achieve the effect of inhibiting osteoclast differentiation and high productivity

Active Publication Date: 2022-07-01
SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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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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  • A stapled peptide that inhibits osteoclast differentiation and its preparation method and application
  • A stapled peptide that inhibits osteoclast differentiation and its preparation method and application
  • A stapled peptide that inhibits osteoclast differentiation and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] Example 1 Preparation of stapled peptides of the present invention that inhibit osteoclast differentiation

[0067] 1. Synthesis of stapled peptides

[0068] like figure 2 shown:

[0069] (1) Preparation of compound 1

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

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

[0072] (2) Preparation of compound 2

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

Embodiment 2

[0086] Identification and structural analysis of the product of Example 2

[0087] The product obtained in step 2 of Example 1 was identified by HPLC and HR-Q-TOF-MS (high resolution matrix-assisted laser desorption ionization time-of-flight mass spectrometry) was used for structural analysis, and the chromatographic mobile phases were acetonitrile and water. Mobile phase A is an aqueous solution with a volume fraction of 0.1% TFA, 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 ; Detection wavelength 214nm and 254nm, injection volume 20μl. It is determined that the peak time of the main peak of the crude product is consistent, and the purity of the stapled 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: The formation of osteoclasts was quantitatively detected 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). Change fresh medium every 2 days. After 5 days of osteoclast induction, the cells were washed twice with PBS, fixed in 4% paraformaldehyde (pH 7.4) for 10 min at room temperature, and stained with TRAP staining kit. Positive multinucleated osteoclasts were counted under light microscope, and the percentage of osteoclasts per well was measured using Image J software.

[0090] see the results Figure 13-14 , the stapled 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 promin...

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Abstract

The present invention relates to a staple peptide for inhibiting osteoclast differentiation, a preparation method and application thereof. The present invention takes amino resin as carrier, and follows template FRATtide: Ac-DPHRLLQQLVLSGNLIKEAVRRLHSR-NH 2 The amino acid sequence was synthesized in the DIC-Oxime condensation system by Fmoc solid-phase synthesis method, and the peptide chain was synthesized on the basis of retaining key amino acid residues. 5 In place of the original amino acid, the linear peptide linked to the resin is subjected to olefin metathesis reaction and cyclization in the dichloroethane solution of Grubbs I reagent, and then cleaved from the resin to obtain the target stapled peptide. The method of the invention is simple and feasible, has high purity and high yield. Further experiments confirmed that the stapled peptide of the present invention can significantly inhibit the differentiation of osteoclasts, and has potential application value in the treatment of osteoporosis and other related diseases.

Description

technical field [0001] The invention belongs to the field of polypeptide medicines, and in particular relates to a stapled peptide for inhibiting osteoclast differentiation and a preparation method and application thereof. 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 easy fracture. Its pathophysiological basis is the imbalance between bone resorption and bone formation. This balance is regulated by osteogenesis-promoting osteoblasts and bone-resorbing osteoclasts. Currently, the most widely used anti-osteoporosis drugs mainly 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, the regulation of important signaling pathways related to the differentiation of...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K7/08C07K1/06C07K1/04C12N5/078A61K38/10A61P19/10
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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