The Split Luciferin Reaction:From Bioorthogonal Chemistry to Bioluminescence Imaging

Studying biological processes on the level of live cells with the help of biocompatible reac-tions has tremendously advanced our understanding of basic biology. However, the great complexity of many human pathologies such as cancer, diabetes and neurodegenerative diseases requires new tools that would allow investigation of biological processes throughout the organism. The 2-cyanobenzothiazole (CBT)-based ligation reaction has received a recent interest in the chemical biology community. It has been reported in the literature for various applications, ranging from fluorescent labelling of proteins to nanostructures formation, and, most importantly, the reaction was shown to proceed in cells. This selective reaction between D-cysteine and hydroxy-CBT (HO-CBT) or amino-CBT (H2N-CBT), also named as split luciferin reaction, generates as product a D-luciferin analogue, one of the most commonly used substrates for bioluminescence imaging (BLI). Therefore, the split luciferin reaction has high potential for BLI applications. In this work, we have shown that production of a luciferin substrate via the split luciferin reaction can be visualized in live mice using BLI. Furthermore, the split luciferin approach allows interrogation of target tissues using a masking approach, where D-luciferin is formed only under certain conditions. This reaction was successfully applied to real-time non-invasive imaging of apoptosis, associated with caspase 3/7 activity. Caspase-dependent release of free D-cysteine from a caspase 3/7 specific peptide substrate allowed selective reaction with H2N-CBT in vivo to form 6-amino-D-luciferin with subsequent light emission in the presence of the firefly luciferase enzyme. Importantly, this strategy was found to be superior to the use of the commercially available DEVD-aminoluciferin substrate for imaging caspase 3/7 activity. The same methodology was extended to imaging activity of other caspases as well as thrombin enzyme in an in vitro set-up. Furthermore, the split luciferin approach enables dual imaging, where each reaction partner would be individually caged to report on separate biological events. This approach was used for simultaneous imaging of caspase 3 and β-galactosidase in vitro, validating the use of the split luciferin reaction for imaging multiple processes. Moreover, the split luciferin reaction was also successfully applied to both quantification of Neutrophil Elastase activity in vitro and real-time non-invasive imaging of Neutrophil Elastase in an in vivo inflammation model. Altogether, the present study suggests that the split luciferin approach is an efficient and versatile tool for in vivo applications

Multiplane 3D superresolution optical fluctuation imaging

By switching fluorophores on and off in either a deterministic or a stochastic manner, superresolution microscopy has enabled the imaging of biological structures at resolutions well beyond the diffraction limit. Superresolution optical fluctuation imaging (SOFI) provides an elegant way of overcoming the diffraction limit in all three spatial dimensions by computing higher-order cumulants of image sequences of blinking fluorophores acquired with a conventional widefield microscope. So far, three-dimensional (3D) SOFI has only been demonstrated by sequential imaging of multiple depth positions. Here we introduce a versatile imaging scheme which allows for the simultaneous acquisition of multiple focal planes. Using 3D cross-cumulants, we show that the depth sampling can be increased. Consequently, the simultaneous acquisition of multiple focal planes reduces the acquisition time and hence the photo-bleaching of fluorescent markers. We demonstrate multiplane 3D SOFI by imaging the mitochondria network in fixed C2C12 cells over a total volume of $65\times65\times3.5 \mu\textrm{m}^3$ without depth scanning.Comment: 7 pages, 3 figure

Development of a bioluminescent nitroreductase probe for preclinical imaging

Bacterial nitroreductases (NTRs) have been widely utilized in the development of novel antibiotics, degradation of pollutants, and gene-directed enzyme prodrug therapy (GDEPT) of cancer that reached clinical trials. In case of GDEPT, since NTR is not naturally present in mammalian cells, the prodrug is activated selectively in NTR-transformed cancer cells, allowing high efficiency treatment of tumors. Currently, no bioluminescent probes exist for sensitive, non-invasive imaging of NTR expression. We therefore developed a "NTR caged luciferin" (NCL) probe that is selectively reduced by NTR, producing light proportional to the NTR activity. Here we report successful application of this probe for imaging of NTR in vitro, in bacteria and cancer cells, as well as in vivo in mouse models of bacterial infection and NTR-expressing tumor xenografts. This novel tool should significantly accelerate the development of cancer therapy approaches based on GDEPT and other fields where NTR expression is important.publishedVersio

Portable bioluminescence systems and methods for in vivo monitoring of molecular and metabolic events in animals

A system for monitoring biological processes in vivo is provided. The system comprises an implantable luciferase biosensor comprising luciferase in a biocompatible matrix and a caged luciferin probe. The caged luciferin probe can be administered to a living subject and upon encountering a biological activity, e.g., enzyme activity, the caged luciferin probe releases free luciferin which can then interact with the biosensor luciferase to generate light. The light can then be detected and/or measured by a light detector. Compositions, methods and kits related to the system are provided herein

Cross coupling of non-activated alkyl halides with alkynyl Grignard reagents catalyzed by a well-defined Ni pincer complex

The nickel pincer complex 1 catalyzes the cross-coupling of the title compounds with remarkable substrate scope and functional group tolerance. A nickel/alkynyl species was isolated and shown to be catalytically competent. THF=tetrahydrofuran, O-TMEDA=bis[2-(N,N-dimethylaminoethyl)] ether

Bioluminescence imaging of small biomolecules

The invention relates to a technique to detect small molecules using Bioluminescence imaging (BLI) to image and quantify non-invasively, in vitro and in vivo,intracellular metabolite fluxes and which can be applied to azido-modified compounds, such as azido-modified biomolecules

A biocompatible "split luciferin" reaction and its application for non-invasive bioluminescent imaging of protease activity in living animals

The great complexity of many human pathologies, such as cancer, diabetes, and neurodegenerative diseases, requires new tools for studies of biological processes on the whole organism level. The discovery of novel biocompatible reactions has tremendously advanced our understanding of basic biology; however, no efficient tools exist for real-time non-invasive imaging of many human proteases that play very important roles in multiple human disorders. We recently reported that the "split luciferin" biocompatible reaction represents a valuable tool for evaluation of protease activity directly in living animals using bioluminescence imaging (BLI). Since BLI is the most sensitive in vivo imaging modality known to date, this method can be widely applied for the evaluation of the activity of multiple proteases, as well as identification of their new peptide-specific substrates. In this unit, we describe several applications of this "split luciferin" reaction for quantification of protease activities in test tube assays and living animals

Development of a Bioluminescent Nitroreductase Probe for Preclinical Imaging.

Bacterial nitroreductases (NTRs) have been widely utilized in the development of novel antibiotics, degradation of pollutants, and gene-directed enzyme prodrug therapy (GDEPT) of cancer that reached clinical trials. In case of GDEPT, since NTR is not naturally present in mammalian cells, the prodrug is activated selectively in NTR-transformed cancer cells, allowing high efficiency treatment of tumors. Currently, no bioluminescent probes exist for sensitive, non-invasive imaging of NTR expression. We therefore developed a "NTR caged luciferin" (NCL) probe that is selectively reduced by NTR, producing light proportional to the NTR activity. Here we report successful application of this probe for imaging of NTR in vitro, in bacteria and cancer cells, as well as in vivo in mouse models of bacterial infection and NTR-expressing tumor xenografts. This novel tool should significantly accelerate the development of cancer therapy approaches based on GDEPT and other fields where NTR expression is important

Discovery of a Potent and Selective Covalent Inhibitor and Activity-Based Probe for the Deubiquitylating Enzyme UCHL1, with Anti-Fibrotic Activity

Ubiquitin carboxy-terminal hydrolase L1 (UCHL1) is a deubiquitylating enzyme which is proposed as a potential therapeutic target in neurodegeneration, cancer, and liver and lung fibrosis. Herein we report the discovery of the most potent and selective UCHL1 probe (IMP-1710) to date based on a covalent inhibitor scaffold and apply this probe to identify and quantify target proteins in intact human cells. IMP-1710 stereoselectively labels the catalytic cysteine of UCHL1 at low nanomolar concentration in cells, and we show that a previously claimed UCHL1 inhibitor (LDN-57444) fails to engage UCHL1 in cells. We further demonstrate that potent UCHL1 inhibitors block pro-fibrotic responses in a cellular model of idiopathic pulmonary fibrosis, supporting a potential therapeutic role for UCHL1 inhibition and providing a basis for future therapeutic development of selective UCHL1 inhibitors