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Updated: Jul 29, 2025
High-Throughput Cellular Profiling of Targeted Protein Degradation Compounds Using HiBiT CRISPR Cell Lines
Published on: November 9, 2020
Discovery of intracellular self-assembly protein degraders driven by tumor-specific activatable bioorthogonal
Ru Si1, Ping Hai2, Yongbiao Zheng2
1School of Pharmacy, Health Science Center, Xi'an Jiaotong University, Xi'an, 710061, China.
Abstract:
Proteolysis Targeting Chimera (PROTAC) is a type of bifunctional chimeric molecule that can directly degrade the binding proteins through the ubiquitin-proteasome pathway. PROTAC has shown great potential in overcoming drug resistance and targeting undruggable targets. However, there are still many shortcomings that need to be solved urgently, including worse membrane permeability and bioavailability induced by their large molecular weight. Herein, we used intracellular self-assembly strategy to construct tumor-specific PROTACs via small molecular precursors. We developed two types of precursors incorporated with azide and alkyne as biorthogonal groups, respectively. These small precursors with improved membrane permeability could react facilely with each other under the catalysis of copper ions with high concentration in tumor tissues, affording novel PROTACs. These novel intracellular self-assembled PROTACs could effectly induce degradation of VEGFR-2 and EphB4 in U87 cells. Meanwhile, they could also promote apoptosis and block cells in S phase. These tumor-specific intracellular self-assembled PROTACs exhibited high selectivity due to the high concentration of copper content in tumor tissue. Moreover, this new strategy could reduce the molecular weight of PROTACs, as well as improve the membrane permeability. These results will greatly expand the applications of bioorthogonal reaction in discovery of novel PROTACs.
Insights
This study introduces novel, small-molecule Proteolysis Targeting Chimeras (PROTACs) that self-assemble inside tumor cells. This strategy improves membrane permeability and targets specific proteins, offering a new approach for cancer therapy.
Area of Science:
- Biochemistry
- Molecular Biology
- Drug Discovery
Background:
- Proteolysis Targeting Chimeras (PROTACs) offer promise for drug resistance and undruggable targets.
- Current PROTACs face challenges with large molecular weight, impacting membrane permeability and bioavailability.
- Targeted protein degradation is a key therapeutic strategy in oncology.
Purpose of the Study:
- To develop tumor-specific PROTACs with enhanced membrane permeability and bioavailability.
- To utilize an intracellular self-assembly strategy for novel PROTAC construction.
- To overcome limitations of traditional PROTACs through small molecular precursors and bioorthogonal reactions.
Main Methods:
- Designed small molecular precursors with azide and alkyne for biorthogonal reactions.
- Utilized intracellular self-assembly catalyzed by copper ions in tumor tissues.
- Assessed PROTAC efficacy in degrading VEGFR-2 and EphB4 in U87 cells.
Main Results:
- Successfully constructed novel, intracellular self-assembled PROTACs.
- Demonstrated effective degradation of VEGFR-2 and EphB4, promoting apoptosis and S-phase cell cycle arrest.
- Achieved tumor-specific selectivity due to high copper ion concentration in tumor tissues.
- Improved membrane permeability and reduced molecular weight compared to conventional PROTACs.
Conclusions:
- Intracellular self-assembly of small PROTAC precursors is a viable strategy for enhanced drug delivery and efficacy.
- This approach offers improved selectivity and overcomes key limitations of traditional PROTACs.
- Bioorthogonal reactions provide a powerful tool for developing next-generation targeted protein degraders.
