PUBLICATION
Target protein identification in live cells and organisms with a non-diffusive proximity tagging system
- Authors
- Sun, Y., Li, C., Deng, X., Li, W., Deng, X., Ge, W., Shi, M., Guo, Y., Yu, Y.V., Zhou, H.B., Jin, Y.N.
- ID
- ZDB-PUB-250109-77
- Date
- 2024
- Source
- eLIFE 13: (Journal)
- Registered Authors
- Guo, Ying
- Keywords
- PafA, biochemistry, chemical biology, chloroquine, dasatinib, proximity labeling, target identification, zebrafish
- MeSH Terms
-
- Humans
- Drug Discovery/methods
- Zebrafish*
- Dasatinib/pharmacology
- Animals
- PubMed
- 39728918 Full text @ Elife
Citation
Sun, Y., Li, C., Deng, X., Li, W., Deng, X., Ge, W., Shi, M., Guo, Y., Yu, Y.V., Zhou, H.B., Jin, Y.N. (2024) Target protein identification in live cells and organisms with a non-diffusive proximity tagging system. eLIFE. 13:.
Abstract
Identifying target proteins for bioactive molecules is essential for understanding their mechanisms, developing improved derivatives, and minimizing off-target effects. Despite advances in target identification (target-ID) technologies, significant challenges remain, impeding drug development. Most target-ID methods use cell lysates, but maintaining an intact cellular context is vital for capturing specific drug-protein interactions, such as those with transient protein complexes and membrane-associated proteins. To address these limitations, we developed POST-IT (Pup-On-target for Small molecule Target Identification Technology), a non-diffusive proximity tagging system for live cells, orthogonal to the eukaryotic system. POST-IT utilizes an engineered fusion of proteasomal accessory factor A and HaloTag to transfer Pup to proximal proteins upon directly binding to the small molecule. After significant optimization to eliminate self-pupylation and polypupylation, minimize depupylation, and optimize chemical linkers, POST-IT successfully identified known targets and discovered a new binder, SEPHS2, for dasatinib, and VPS37C as a new target for hydroxychloroquine, enhancing our understanding these drugs' mechanisms of action. Furthermore, we demonstrated the application of POST-IT in live zebrafish embryos, highlighting its potential for broad biological research and drug development.
Genes / Markers
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Orthology
Engineered Foreign Genes
Mapping