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bioRxiv · 10.1101/2024.09.06.611731

Target protein identification in live cells and organisms with a non-diffusive proximity tagging system

Sun, Y.·Li, C.·Deng, X.·Li, W.·Ge, W.·Shi, M.·Guo, Y.·Yu, Y. V.·Zhou, H.-b.·Jin, Y. N.
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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 (PafA) 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.

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Sun, Y., Li, C., Deng, X., Li, W., Ge, W., Shi, M., Guo, Y., Yu, Y. V., Zhou, H.-b., Jin, Y. N.. 2024-09-08. Target protein identification in live cells and organisms with a non-diffusive proximity tagging system. https://doi.org/10.1101/2024.09.06.611731

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