Iterative design leads to a smart probe capable of quantifying autophagic flux with switchable fluorescence via engaging MAP1LC3/LC3

  • Autophagy. 2026 May 20:1-18. doi: 10.1080/15548627.2026.2674717.
Yaping Lu  1 ,  Ning Wang  2 ,  Meihui Liu  1 ,  Zhepei Lu  2 ,  Shiqi Fan  2 ,  Weisong Lv  2 ,  Yingmei Lu  1  3 ,  Feng Han  4  5  6  7 ,  Xin Li  2
Affiliations
  • 1. Collaborative Innovation Center for Cardiovascular Disease Translational Medicine, Key Laboratory of Modern Toxicology of Ministry of Education, School of Basic Medical Sciences, Nanjing Medical University, Nanjing, China.
  • 2. State Key Laboratory of Chinese Medicine Modernization, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, China.
  • 3. Department of Nephrology, The First Affiliated Hospital of Nanjing Medical University, Nanjing, China.
  • 4. Medical Basic Research Innovation Center for Cardiovascular and Cerebrovascular Diseases, Ministry of Education, Key Laboratory of Cardiovascular & Cerebrovascular Medicine, School of Pharmacy, Nanjing Medical University, Nanjing, China.
  • 5. Gusu School, Nanjing Medical University, Suzhou Municipal Hospital, The Affiliated Suzhou Hospital of Nanjing Medical University, Suzhou, China.
  • 6. Northern Jiangsu Institute of Clinical Medicine, The Affiliated Huaian No.1 People's Hospital of Nanjing Medical University, Huaian, China.
  • 7. Jiangsu Province Innovation Center for Brain-Inspired Intelligence Technology, Nanjing, China.
PMID: 42163447 DOI: 10.1080/15548627.2026.2674717
Abstract

Tracking macroautophagic/autophagic flux in live cells is vital for understanding its pathophysiology; however, its dynamic nature complicates assay development. Although fluorescent protein-tagged markers and microenvironment-sensitive small-molecule fluorescent probes have been developed, non-transfection-based and highly specific assays remain underexplored. In this study, we present the design, synthesis, and application of an activity-based Autophagy probe (ATP1) for dynamically quantifying autophagic flux. ATP1 was developed through an iterative, docking-guided design strategy to secure MAP1LC3/LC3 engagement, coupled with in-depth analysis of structure-fluorescence relationships to program dual smart-signal behaviors. It displays LC3-binding-triggered fluorogenic activation and autophagosome-lysosome fusion-triggered ratiometric changes. By engaging LC3, the probe is inherently specific to Autophagy, and its dynamic signal enables real-time tracking of autophagic flux with high sensitivity. We demonstrate ATP1's exceptional performance in live cells and mice, with a dynamic signal paralleling the mRFP-GFP-LC3 assay. Notably, ATP1 provides significant benefits, including low background signals, compatibility with primary cells, and effectiveness in wild-type mice, where transfection-based assays are often impractical. Furthermore, the probe aids in the discovery of Autophagy modulators. In conclusion, ATP1 offers a straightforward, specific, and non-transfection-based method for assessing autophagic flux, serving as a powerful tool for advancing Autophagy research.Abbreviations: 3-MA: 3-methyladenine; ATP: Autophagy probe; BafA1: bafilomycin A1; CBF: cerebral blood flow; FP: fluorescent protein; HBMECs: human brain microvascular endothelial cells; HBSS: Hanks' balanced salt solution; HEPES: 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid; ITC: isothermal titration calorimetry; LIR: LC3-interacting region; LSCI: laser speckle contrast imaging; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MFI: mean fluorescence intensity; OGD: oxygen-glucose deprivation; PBS: phosphate-buffered saline; Rapa: rapamycin; Wort: wortmannin.

Keywords
Autolysosome; autophagosome; autophagy imaging; fluorescent probe; microtubule-associated protein 1 light chain 3; ratiometric signal.
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