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

Activity-based protein profiling reveals deubiquitinase and aldehyde dehydrogenase targets of a cyanopyrrolidine probe

Ubiquitin carboxy-terminal hydrolase L1 (UCHL1), a deubiquitinating enzyme (DUB), is a potential drug target in various cancers, and liver and lung fibrosis. However, bona fide functions and substrates of UCHL1 remain poorly understood. Herein, we report the characterization of UCHL1 covalent inhibitor MT16-001 based on a thiazole cyanopyrrolidine scaffold. In combination with chemical proteomics, a closely related activity-based probe (MT16-205) was used to generate a comprehensive quantitative profile for on- and off-targets at endogenous cellular abundance. Both compounds are selective for UCHL1 over other DUBs in intact cells but also engage a range of other targets with good selectivity over the wider proteome, including aldehyde dehydrogenases, redox-sensitive Parkinson’s disease related protein PARK7, and glutamine amidotransferase. Taken together, these results underline the importance of robust profiling of activity-based probes as chemical tools and highlight the cyanopyrrolidine warhead as a versatile platform for liganding diverse classes of protein with reactive cysteine residues which can be used for further inhibitor screening, and as a starting point for inhibitor development

Early-Stage Incorporation Strategy for Regioselective Labeling of Peptides using the 2-Cyanobenzothiazole/1,2-Aminothiol Bioorthogonal Click Reaction

Herein, we describe a synthetic strategy for the regioselective labeling of peptides by using a bioorthogonal click reaction between 2‐cyanobenzothiazole (CBT) and a 1,2‐aminothiol moiety. This methodology allows for the facile and site‐specific modification of peptides with various imaging agents, including fluorophores and radioisotope‐containing prosthetic groups. We investigated the feasibility of an early‐stage incorporation of dipeptide 1 into targeting vectors, such as c[RGDyK(C)] and HER2 pep, during solid‐phase peptide synthesis. Then, the utility of the click reaction to label bioactive peptides with a CBT‐modified imaging agent (FITC–CBT, 9) was assessed. The ligation reaction was found to be highly selective and efficient under various conditions. The fluorescently labeled peptides 2 and 3 were obtained in respective yields of 88 and 82 % under optimized conditions.We gratefully acknowledge the Leenaards Foundation (grant # 3699) and NSERC for financial support

Overview on Multienzymatic Cascades for the Production of Non-canonical α-Amino Acids

SM-R thanks the University of Granada for the support provided by project PPJI2017-1 and the European Cooperation in Science and Technology (COST Action CA15133). Authors are also grateful to the Andalusian Regional Government through Endocrinology & Metabolism Group (CTS-202).The 22 genetically encoded amino acids (AAs) present in proteins (the 20 standard AAs together with selenocysteine and pyrrolysine), are commonly referred as proteinogenic AAs in the literature due to their appearance in ribosome-synthetized polypeptides. Beyond the borders of this key set of compounds, the rest of AAs are generally named imprecisely as non-proteinogenic AAs, even when they can also appear in polypeptide chains as a result of post-transductional machinery. Besides their importance as metabolites in life, many of D-α- and L-α-“non-canonical” amino acids (NcAAs) are of interest in the biotechnological and biomedical fields. They have found numerous applications in the discovery of new medicines and antibiotics, drug synthesis, cosmetic, and nutritional compounds, or in the improvement of protein and peptide pharmaceuticals. In addition to the numerous studies dealing with the asymmetric synthesis of NcAAs, many different enzymatic pathways have been reported in the literature allowing for the biosynthesis of NcAAs. Due to the huge heterogeneity of this group of molecules, this review is devoted to provide an overview on different established multienzymatic cascades for the production of non-canonical D-α- and L-α-AAs, supplying neophyte and experienced professionals in this field with different illustrative examples in the literature. Whereas the discovery of new or newly designed enzymes is of great interest, dusting off previous enzymatic methodologies by a “back and to the future” strategy might accelerate the implementation of new or improved multienzymatic cascades.University of Granada PPJI2017-1European Cooperation in Science and Technology (COST) CA15133Andalusian Regional Government through Endocrinology & Metabolism Group CTS-20

Precursor molecule for the synthesis of d-luciferin

The present invention relates to precursor molecules for the synthesis of D-luciferin, or blocked D-luciferin and derivatives thereof, which are functionalized in different positions. Further, the use of functionalized precursor molecules for the synthesis of D-luciferin or blocked D-luciferin and methods of synthesising blocked D-luciferin and D-luciferin or derivatives thereof are described. Also described is a kit of parts for screening assay comprising said functionalized precursor molecules to D-luciferin or blocked D-luciferin and derivatives thereof and the uses of such a screening assay for the detection of molecules for the study of molecular uptake and for the detection of a reducing environment. Methods of detection of bio- molecules such as metabolites, activity of enzymes and proteases and methods for determining a sequence within a peptide of protein cleaved by specific enzymes are described. Also a method of determining the cysteine and derivatives thereof concentration in a cell or tissue sample is described. Precursor molecules of D-luciferin according to the invention are advantageous as they are small and travel easily around live systems and allow tissue cells or organs to be targeted specifically. Further, the methods and screening assays described allow the simultaneous study of multiple biological processes described above in one experiment only

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