Atg4B and Cathepsin B-Triggered in Situ Luciferin Formation for Precise Cancer Autophagy Bioluminescence Imaging

Abstract

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 Dcysteine 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.

Protease-Responsive Bitext Editing Biopolymer for Breast Cancer Imaging In Vivo

“…Contrarily, the fluorescence of CA-074-Me-pretreated group presents little recovery, thereby indicating that the CatB cuts the peptide substrate probes at a specific cleavage site (Arg-Arg-) to trigger the fluorescence recovery (Figures b and S4). − We have investigated the optimal inhibitor concentration considering the inhibition effect and cost saving. CA-074-Me (CatB inhibitor) concentration of 5 mM was chosen as the optimal working concentration in the subsequent experiments in vitro (Figure S5).…”