Lysosome-targeted fluorescence probe for discriminating cysteine from homocysteine/glutathione and assessing autophagy in drug-induced liver injury

Abstract

Autophagy inhibition is an early cause of drug-induced liver injury (DILI). Cysteine (Cys) levels in lysosomes are associated with autophagy. Detecting Cys in lysosomes visually is a new requirement for DILI early diagnosis and efficacy assessment. Hence, the development of excellent fluorescent probes that can detect Cys in lysosomes with reliability, selectivity, and sensitivity is needed. In this work, a lysosome-targeted fluorescent probe LTCP for discriminating Cys from Hcy/GSH has been developed based on the sulfonylbenzoxadiazole (SBD) chromophore containing a morpholine moiety. Encounter of LTCP with Cys will trigger a sequential aromatic substitution-rearrangement reaction yielding an amino SBD derivative product and generate an obvious orange response fluorescence (λex/λem: 440/583 nm), while encounter with Hcy/GSH forms an alksulfydryl SBD derivative product via one-step aromatic substitution reaction and elicits a clear different green fluorescence (λex/λem: 400/520 nm). With the help of confocal fluorescence imaging using probe LTCP, an increase of lysosomal Cys level is observed after Rapa activates autophagy, whereas Cys concentration decreases after 3-MA inhibits autophagy in HepG2 cells. Additionally, LTCP was successfully applied for high-fidelity imaging of Cys changes in autophagy during liver injury induced by acetaminophen (APAP).