Autophagy plays a
crucial role in tumorigenesis and progression,
but current approaches to visualize it in vivo show limited precision
due to their single-analyte-responsive mode. Hence, by simultaneously
employing dual autophagy enzymes Atg4B and cathepsin B to trigger
the in situ formation of luciferin, we herein propose a strategy for
precise autophagy bioluminescence imaging. An Atg4B-responsive peptide
Ac-Thr-Phe-Gly-d-Cys (TFGC) and a cathepsin B-activatable
compound Ac-Lys-Gly-Arg-Arg-CBT (KGRR-CBT) were rationally designed.
During tumor autophagy, these two compounds were uptaken by cancer
cells and cleaved by their corresponding enzymes to yield d-cysteine and 2-cyano-6-aminobenzothiazole, respectively, which underwent
a CBT-Cys click reaction to yield d-aminoluciferin, turning
the bioluminescence “on”. The responsiveness of these
two compounds toward the two enzymes was tested in vitro, and the
ability to turn bioluminescence “on” was validated in
living cancer cells and in vivo. We anticipate that our precise autophagy
imaging strategy could be further applied for the diagnosis of autophagy-related
diseases in the near future.