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.
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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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