A Biocompatible in Vivo Ligation Reaction and Its Application for Noninvasive Bioluminescent Imaging of Protease Activity in Living Mice

Abstract

The discovery of biocompatible reactions has had a tremendous impact on chemical biology, allowing the study of numerous biological processes directly in complex systems. However, despite the fact that multiple biocompatible reactions have been developed in the past decade, very few work well in living mice. Here we report that D-cysteine and 2-cyanobenzothiazoles can selectively react with each other in vivo to generate a luciferin substrate for firefly luciferase. The success of this “split luciferin” ligation reaction has important implications for both in vivo imaging and biocompatible labeling strategies. First, the production of a luciferin substrate can be visualized in a live mouse by bioluminescence imaging (BLI), and furthermore allows interrogation of targeted tissues using a “caged” luciferin approach. We therefore applied this reaction to the real-time non-invasive imaging of apoptosis associated with caspase 3/7. Caspase-dependent release of free D-cysteine from the caspase 3/7 peptide substrate Asp-Glu-Val-Asp-D-Cys (DEVD-(D-Cys)) allowed selective reaction with 6-amino-2-cyanobenzothiazole (NH2-CBT) in vivo to form 6-amino-D-luciferin with subsequent light emission from luciferase. Importantly, this strategy was found to be superior to the commercially-available DEVD-aminoluciferin substrate for imaging of caspase 3/7 activity. Moreover, the split luciferin approach enables the modular construction of bioluminogenic sensors, where either or both reaction partners could be caged to report on multiple biological events. Lastly, the luciferin ligation reaction is three orders of magnitude faster than Staudinger ligation suggesting further applications for both bioluminescence and specific molecular targeting in vivo.

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.…”

A Biocompatible “Split Luciferin” Reaction and Its Application for Non‐Invasive Bioluminescent Imaging of Protease Activity in Living Animals

“…We previously reported that D-cysteine and 2-cyanobenzothiazole (CBT) can selectively react with each other in vivo to generate luciferin substrates for firefly luciferase (“split luciferin reaction”, Fig. 1) (Godinat et al, 2013). …”

“…We previously reported application of this “split luciferin reaction” for the real-time and non-invasive imaging of apoptosis, associated with activation of caspase 3/7 (Godinat et al, 2013). Caspase-dependent release of free D-cysteine from the caspase 3/7-specific peptidic substrate Asp-Glu-Val-Asp-D-Cys (DEVD-(D-Cys)) allowed further reaction with 6-amino-2-cyanobenzothiazole (NH 2 -CBT) in vivo and subsequent formation of 6-amino-D-luciferin.…”

“…Since this compound is known to be an excellent substrate for luciferase enzyme, its formation leads to the generation of light that is proportional to the activity of caspase 3/7. Importantly, this “split luciferin” strategy was found to be more sensitive when compared to the commercially available DEVD-aminoluciferin substrate (Godinat et al, 2013), in which the entire luciferin is caged with the same peptidic sequence. The underlining principle of the use of “split luciferin reaction” for proteases imaging is depicted in Fig.…”