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Application of Natural Small Molecular Compounds in Inhibiting Ubiquitin Chain Synthesis

A technology for small molecule compounds and synthesis reactions, applied in the fields of biotechnology and medicine, it can solve the problems of unsatisfactory specificity, high cytotoxicity, and many cell signaling pathways, and achieve the effect of good specificity, low IC50 value and high efficacy.

Active Publication Date: 2021-08-31
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] Currently, only the proteasome inhibitor PS34 (Valcade) and the E3 ubiquitin ligase CRL4CRBN inhibitor lenalidomide or thalidomide have entered clinical application, but these drugs affect many cell signaling pathways, the specificity is not ideal, and the cytotoxicity is relatively high

Method used

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  • Application of Natural Small Molecular Compounds in Inhibiting Ubiquitin Chain Synthesis
  • Application of Natural Small Molecular Compounds in Inhibiting Ubiquitin Chain Synthesis
  • Application of Natural Small Molecular Compounds in Inhibiting Ubiquitin Chain Synthesis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0073] Preparation of each raw material in embodiment 1 ubiquitin chain synthesis reaction

[0074] 1. Purification of ubiquitin (Ub)

[0075] Untagged ubiquitin cDNA was constructed on the prokaryotic expression vector pET14b. transforming Escherichia coli

[0076] (BL21(DE3) / pJY2 strain), expression was induced with 0.5 mM IPTG for 4 hours at 37°C. After collecting 1L of Escherichia coli and discarding the supernatant, resuspend with 20ml of buffer solution (50mM Tris-Cl pH7.6, 1mM PMSF, 0.02% (v / v) NP-40, 0.4mg / ml lysozyme), and carry out Sonication. After centrifugation at 20,000g for 20 minutes, transfer the supernatant to a clean container, and slowly add 70% perchloric acid dropwise while stirring at 4°C. At this time, most of the miscellaneous proteins will denature and precipitate, and continue stirring when the pH reaches about 4.0. After 30 minutes, centrifuge at 20000 g for 20 minutes, and take the supernatant. Dialyze twice against 100 volumes of pH 4.5 ammon...

Embodiment 2

[0085] 1. Ubiquitin chain synthesis reaction

[0086] Prepare the following reaction system:

[0087]

[0088]A control group and 11 sample groups were set; wherein, the control group also contained 5% DMSO, and the sample group also contained natural small molecule compounds (purchased from Selleck, dissolved in DMSO), and the natural small molecule compounds were quercetin ( Sample group 1), luteolin (sample group 2), myricetin (sample group 3), fisetin (sample group 4), morin (sample group 5), kaempferol (sample group 6), apigenin ( Sample group 7), anthocyanin (sample group 8), buterin (sample group 9), coreopsis chalcone (sample group 10) and epigallocatechin gallate (sample group 11).

[0089] After the sample group and the control group were reacted at 30°C for 30 minutes, SDS-PAGE sample buffer was added to terminate the reaction, and then electrophoresis and Western Blot were performed to detect ubiquitin protein. The detection results were as follows: figure 1 s...

Embodiment 3

[0097] 1. The experiment of synthesizing ubiquitin chains by using in vitro ubiquitin aminolysis reaction shows that natural small molecule compounds inhibit the enzymatic activity of Ubc13

[0098] Prepare the following reaction system:

[0099]

[0100] Control group and sample group are set; Wherein, control group also contains 5% DMSO, and sample group also contains natural small molecule compound (dissolved in DMSO), and this natural small molecule compound is quercetin (sample group 1), luteolin (sample group 2), epigallocatechin gallate (sample group 3). The same group of samples was set under two conditions of adding or not adding 10nM TRAF6 protein, and placed at 30°C for a reaction ranging from 5 minutes to 4 hours. After the reaction products were separated by SDS-PAGE electrophoresis, the ubiquitin protein was detected by WesternBlot. Results such as Figure 9 ~ Figure 11 shown.

[0101] Depend on Figures 9 to 11 As shown, it shows that dendrobetin, luteoli...

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Abstract

The invention discloses the application of a natural small molecular compound in inhibiting the synthetic reaction of ubiquitin chains. The present invention finds for the first time that a natural small molecule compound with a specific structure can inhibit the synthesis reaction of ubiquitin chains. The natural small molecule compound has the characteristics of low IC50 value, high efficacy and good specificity; in addition, the present invention also resolves ubiquitin step by step in vitro The experiments of ylation reaction and intracellular ubiquitination reaction proved that natural small molecular compounds can inhibit the activity of ubiquitin-conjugating enzyme Ubc13 and then inhibit the ubiquitin-aminolysis reaction mediated by the conjugating enzyme Ubc13.

Description

technical field [0001] The invention relates to the fields of biotechnology and medicine, in particular to the application of natural small molecular compounds in inhibiting ubiquitin chain synthesis. Background technique [0002] Ubiquitination is caused by the ubiquitin protein itself as a signaling molecule, through the ubiquitin-activating enzyme (Ubiquitin- [0003] The cascade reaction of activating Enzyme) E1, Ubiquitin-conjugating Enzyme (Ubiquitin-conjugating Enzyme) E2 and Ubiquitin Ligase (Ubiquitin Ligase) E3 is covalently modified to the lysine residue of the substrate protein, thereby realizing the ubiquitination of the protein. prime modification. [0004] A single ubiquitin molecule modifies a single lysine residue on a substrate protein, resulting in monoubiquitination. Multiple ubiquitin molecules modify multiple lysine residues on a substrate protein, resulting in polyubiquitination. There are 7 lysine residues in the ubiquitin protein body, K48, K63, K...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K31/352A61K31/12A61K31/353A61P43/00A61P25/16
CPCA61K31/12A61K31/352A61K31/353
Inventor 夏总平李海东戴立思
Owner ZHEJIANG UNIV
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