Stapled peptide conjugate for degrading MDM2/MDMX protein by protein-targeted chimera and application thereof

A technology of conjugates and stapled peptides, applied in the field of polypeptide drugs, can solve the problems of poor membrane permeability, many types of binding proteins and complexities, and achieve the effect of improving stability and high serum stability.

Active Publication Date: 2021-12-28
THE NAVAL MEDICAL UNIV OF PLA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] In the past, the inhibitors targeting p53-MDM2 / MDMX protein were almost all chemical small molecules. Since the binding interface between MDM2 / MDMX and other proteins is large, and the binding proteins are diverse and complex, small molecule inhibitors are difficult to Complete suppression of MDM2 / MDMX functions
However, linear polypeptides have problems such as low stability and poor membrane permeability.

Method used

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  • Stapled peptide conjugate for degrading MDM2/MDMX protein by protein-targeted chimera and application thereof
  • Stapled peptide conjugate for degrading MDM2/MDMX protein by protein-targeted chimera and application thereof
  • Stapled peptide conjugate for degrading MDM2/MDMX protein by protein-targeted chimera and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Example 1: Preparation of a proteolytic targeting chimera staple peptide conjugate

[0057] 1. Synthesis of stapled peptide (taking SPMI-HIF2-1 as an example)

[0058] Synthetic roadmap as figure 2 Shown:

[0059] (1) Preparation of compound 1

[0060] Take amino resin 333mg (sample loading is 0.30mmol·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.

[0061] 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 5 times each.

[0062] (2) Preparation of Compound 2

[0063] Mix the first amino acid in the sequence (1mmol), Oxyme (142mg, 1mmol) and DIC (155.0μL, 1mmol) in 6ml of NMP, the synthesis of PEG and amino acid is the same, add to the resin and shake at 60°C for 20min ( R 8 / S 5 The last amino acid was reacted for 2h), and the resin was washed wi...

Embodiment 2

[0078] Example 2: Liquid phase and mass spectrometry characterization of a proteolytic targeting chimeric staple peptide conjugate

[0079] The product of Example 1 was identified by HPLC and structurally analyzed by HR-Q-TOF-MS, 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% to 80%); flow rate 1mL·min-1; detection wavelength 214nm and 254nm, injection volume 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 conjugate prepared by the present invention is >98% ( image 3 ). The analysis results by HR-Q-TOF-MS mass spectrometer are as follows: Figure 4 shown.

Embodiment 3

[0080] Example 3: Using colon cancer cells to detect the extracellular anti-tumor effect of stapled peptide conjugates

[0081] Colon cancer cell line HCT116 and P53-knockout colon cancer cell line HCT116- / - were cultured in high-sugar DMEM containing 10% fetal bovine serum, 100 U / ml penicillin and 100 mgL-1 streptomycin at 37°C, 5% CO 2 Routine culture and passage in the incubator. Spread 96-well plates with 1000 cells per well, and add different concentrations (0, 3.125, 6.25, 12.5, 25, 50, 100 μm) of polypeptides PMI-HIF-1, PMI-HIF-2, and PMI-HIF the next day -3; SPMI2; SPMI-HIF2-1, SPMI-HIF2-2, SPMI-HIF2-3, after 72h, 10 μL of CCK8 reagent was added to each well, and incubated at 37°C for 1h. The absorbance value (OD) of each well was detected at a wavelength of 450 nm with a microplate reader (BioTek, Vermont, USA), and the cell viability at different concentrations was calculated according to the OD value.

[0082] The result is as Figure 5 The results showed that th...

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Abstract

The invention relates to the field of polypeptide drugs, in particular to a stapled peptide conjugate for degrading MDM2/MDMX protein by protein-targeted chimeras (PROTACTs) and a preparation method and application thereof. The stapled peptide conjugate is composed of three parts: a first peptide fragment suitable for binding with the MDM2/MDMX protein, a second peptide fragment suitable for binding with E3 ubiquitin ligase VHL, and a connecting arm enabling the first peptide fragment and the second peptide fragment to be connected. The E3 ubiquitin ligase VHL can be used for carrying out ubiquitination modification on the MDM2/MDMX protein, so that protease degrades the ubiquitination MDM2/MDMX protein, a P53 factor is activated to play roles in killing cancer cells and inhibiting tumor formation ability, and the E3 ubiquitin ligase VHL has a potential application value.

Description

technical field [0001] The invention relates to the field of polypeptide medicine, in particular to a protein-targeting chimera (PROTACTs) degrading staple peptide conjugate of MDM2 / MDMX protein and its preparation method and application. Background technique [0002] Tumor has become one of the major diseases that are increasingly common and seriously threaten human life and quality of life. Tumor treatment methods will be selected according to the type of tumor and the maturity of the current technology, or a combination of multiple treatments. Current tumor treatment methods include surgery, radiation therapy, and hormone therapy, all of which have side effects to varying degrees. In contrast, drug molecular targeted therapy is preferred. As p53 is the most important tumor suppressor, targeting the interaction between p53 and two key negative regulators MDM2 and MDMX for tumor therapy has become a research hotspot. Therefore, it is considered to be the most direct and ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C07K1/08C07K1/06C07K1/04C07K1/20A61K38/08A61P35/00
CPCC07K7/06A61P35/00C07K2319/70C07K2319/31A61K38/00Y02P20/55
Inventor 李翔陆五元胡宏岗耿晨晨陈思
Owner THE NAVAL MEDICAL UNIV OF PLA
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