Strategy for Dual-Analyte Luciferin Imaging: In Vivo Bioluminescence Detection of Hydrogen Peroxide and Caspase Activity in a Murine Model of Acute Inflammation

Abstract

In vivo molecular imaging holds promise for understanding the underlying mechanisms of health, injury, aging, and disease, as it can detect distinct biochemical processes such as enzymatic activity, reactive small-molecule fluxes, or post-translational modifications. Current imaging techniques often detect only a single biochemical process, but, within whole organisms, multiple types of biochemical events contribute to physiological and pathological phenotypes. In this report, we present a general strategy for dual-analyte detection in living animals that employs in situ formation of firefly luciferin from two complementary caged precursors that can be unmasked by different biochemical processes. To establish this approach, we have developed Peroxy Caged Luciferin-2 (PCL-2), a H2O2-responsive boronic acid probe that releases 6-hydroxy-2-cyanobenzothiazole (HCBT) upon reacting with this reactive oxygen species (ROS), as well as a peptide-based probe, Ile-Glu-Thr-Asp-D-Cys (IETDC) which releases D-cysteine in the presence of active caspase 8. Once released, HCBT and D-cysteine form firefly luciferin in situ, giving rise to a bioluminescent signal if and only if both chemical triggers proceed. This system thus constitutes an AND-type molecular logic gate that reports on the simultaneous presence of H2O2 and caspase 8 activity. Using these probes, chemoselective imaging of either H2O2 or caspase 8 activity was performed in vitro and in vivo. Moreover, concomitant use of PCL-2 and IETDC in vivo establishes a concurrent increase in both H2O2 and caspase 8 activity during acute inflammation in living mice. Taken together, this method offers a potentially powerful new chemical tool for studying simultaneous oxidative stress and inflammation processes in living animals during injury, aging, and disease, as well as a versatile approach for concurrent monitoring of multiple analytes using luciferin-based bioluminescence imaging technologies.

In vivo bioluminescence imaging reveals copper deficiency in a murine model of nonalcoholic fatty liver disease

“…Moreover, impressive advances in luciferase engineering and ease of targeting by genetic encoding permit the broad use of such reporters for in vivo imaging with cell and tissue specificity (41,43,44). We and others have developed caged luciferins, which are enzymeinert luciferin derivatives that are chemically unmasked to the luciferin substrate in the presence of an analyte or biochemical event of interest for subsequent enzymatic generation of light (45)(46)(47)(48)(49)(50); in particular, our laboratory has used this approach to develop bioluminescent H 2 O 2 reporters (45,47). To create a Cu + -responsive luciferin probe, we exploited a Cu + -dependent oxidative cleavage reaction mediated by the tetradentate ligand TPA (Fig.…”

In vivo bioluminescence imaging of labile iron accumulation in a murine model of Acinetobacter baumannii infection

“…Work from our laboratory and others has demonstrated the utility of caged luciferins in vivo (28)(29)(30) for measuring transient small molecules (31)(32)(33)(34), enzyme and transporter activities (34)(35)(36)(37)(38)(39)(40)(41)(42)(43)(44)(45)(46), protein-protein and cell-cell interactions (42,47,48), and copper (49). Indeed, previous work from our laboratory utilized a Cu-dependent oxidation reaction to uncage luciferin for in vivo copper imaging (50), a first demonstration of a general activitybased sensing (ABS) strategy which we envisioned expanding to other essential metals in biology by changing the reaction trigger.…”

Recent progress in luminescent proteins development

“…Split caged D-luciferin can be used to detect two different biochemical processes [34 ]. Peroxy-caged luciferin-2 (PCL-2) releases 6-hydroxy-2-cyanobenzothianole (HCBT) upon reacting with H 2 O 2 .…”

“…Because the luminescence signals are dependent on the above two biochemical processes, this system can be used for detection of both H 2 O 2 and caspase-8 activities. Using this system, endogenous H 2 O 2 and caspase-8 activities were detected in living mice during acute inflammation upon lipopolysaccharide stimulation [34 ].…”