a National Glycoengineering Research Center, Shandong Key Laboratory of Carbohydrate Chemistry and Glycobiology, Shandong University, Qingdao, Shandong, 266237, China;
b School of Life Sciences, Medical Science and Technology Innovation Center, Shandong First Medical University & Shandong Academy of Medical Sciences, Jinan, 250117, China;
c School of Pharmaceutical Sciences, Shandong First Medical University & Shandong Academy of Medical Sciences, Jinan, 250101, China;
d Shandong Academy of Pharmaceutical Sciences, Engineering Research Center for Sugar and Sugar Complex, National-Local Joint Engineering Laboratory of Polysaccharide Drugs, Key Laboratory of Carbohydrate and Glycoconjugate Drugs, Jinan, 250101, China;
e College of Clinical and Basic Medicine (Institute of Basic Medicine), Shandong First Medical University, Jinan, 250101, China;
f Zunyi Medical University, Zunyi, Guizhou, 563006, China;
g Department of General Surgery, The First Affiliated Hospital of Shandong First Medical University & Shandong Academy of Medical Sciences, Jinan, 250014, China;
h School of Health Industry Management & School of Medicine & Health Sciences, University of Sanya, Sanya, Hainan, 572022, China;
i Departments of Diagnostic Radiology, Surgery, Chemical and Biomolecular Engineering, and Biomedical Engineering, Yong Loo Lin School of Medicine and College of Design and Engineering, National University of Singapore, Singapore, 119074, Singapore
Funds:
This work is supported by the Key Technologies Research and Development Program, China (Grant No.: 2021YFC2103100), Beijing Municipal Natural Science FoundationKey Research Project of the Daxing, China (Grant No.: L246029), National Natural Science Foundation of China (Grant Nos.: 82302743 and 22107059), Key Technology and Development Program of Shandong Province, China (Project No.: 2022SFGC0103), Young Elite Scientists Sponsorship Program by China Association for Science and Technology, China (Project No.: CACM-2023-QNRC1-02), Natural Science Foundation of Shandong Province, China (Grant Nos.:ZR2021QH057 and ZR2022QH304), National Administration of Traditional Chinese Medicine-Shandong Province Joint Construction of Traditional Chinese Medicine Technology Project, China (Project No.: GZY-KJS-SD-2023-085), Taishan Scholars Program, China (Project Nos.: TSQN202211221 and TSQN202408252), Shandong Science Fund for Excellent Young Scholars, China (Grant No.: ZR2022YQ66), Shandong Province Traditional Chinese Medicine Science and Technology Project, China (Project No.: Q- 2023059), Shandong Province Medical and Health Science and Technology Project, China (Project No.: 202404070376).
Conventional ex vivo drug screening platforms struggle to recapitulate native subcellular microenvironments, leading to high off-target rates and compromised discovery of bioactive compounds. To address this, we developed subcellular target- tracking fluorescent-visualization-based interaction screening (SubTrack-FVIS), a platform combining super-resolution imaging with target-specific fluorescent tagging. SubTrack-FVIS first maps nanoscale spatial distributions of drug targets within living cells, then screens compound libraries to identify molecules specifically binding to target-enriched domains, and finally quantifies drug-target interactions through super- resolution imaging tracking. Compared to traditional toolbox, SubTrack-FVIS reduces off-target effects by evaluating compound binding within native subcellular architectures. When applied to the lysosomal vacuolar H+-ATPases (V-ATPase) subunit, ATP6V1A, a validated anti-cancer target, this approach identified for lysosomal alkalization fluorescent drug (LAFD) as a potent inhibitor. Super-resolution imaging revealed LAFD's dynamic binding to ATP6V1A clusters, enabling real-time visualization of V-ATPase inhibition and subsequent lysosomal destabilization. Crucially, SubTrack-FVIS uncovered LAFD's unique mechanism of blocking autophagosome-lysosome fusion, resolving autophagic flux obstruction at sub-100 nm resolution. This platform establishes a visualization framework for discovering drugs within physiological subcellular contexts while simultaneously decoding their mechanistic impacts, offering application potential for target-centric drug development.