Department of Neurology, Medical Research Institute, Zhongnan Hospital of Wuhan University, Wuhan University, Wuhan, China; Frontier Science Center for Immunology and Metabolism, Wuhan University, Wuhan, China
Hai-bing Zhou
Department of Hematology, Zhongnan Hospital of Wuhan University, School of Pharmaceutical Sciences, Wuhan University, Wuhan, China; State Key Laboratory of Virology, Hubei Province Engineering and Technology Research Center for Fluorinated Pharmaceuticals, Key Laboratory of Combinatorial Biosynthesis and Drug Discovery (Wuhan University), Ministry of Education, Frontier Science Center for Immunology and Metabolism, Wuhan University, Wuhan, China
Department of Neurology, Medical Research Institute, Zhongnan Hospital of Wuhan University, Wuhan University, Wuhan, China; Frontier Science Center for Immunology and Metabolism, Wuhan University, Wuhan, China
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.
