Luciferin

Summary

Luciferin () is a generic term for the light-emitting compound found in organisms that generate bioluminescence. Luciferins typically undergo an enzyme-catalyzed reaction with molecular oxygen. The resulting transformation, which usually involves splitting off a molecular fragment, produces an excited state intermediate that emits light upon decaying to its ground state. The term may refer to molecules that are substrates for both luciferases and photoproteins. Luciferins are a class of small-molecule substrates that react with oxygen in the presence of a luciferase (an enzyme) to release energy in the form of light. It is not known just how many types of luciferins there are, but some of the better-studied compounds are listed below. Because of the chemical diversity of luciferins, there is no clear unifying mechanism of action, except that all require molecular oxygen, The variety of luciferins and luciferases, their diverse reaction mechanisms and the scattered phylogenetic distribution indicate that many of them have arisen independently in the course of evolution. Firefly luciferin Firefly luciferin is the luciferin found in many Lampyridae species. It is the substrate of beetle luciferases (EC 1.13.12.7) responsible for the characteristic yellow light emission from fireflies, though can cross-react to produce light with related enzymes from non-luminous species. The chemistry is unusual, as adenosine triphosphate (ATP) is required for light emission, in addition to molecular oxygen. Latia luciferin is, in terms of chemistry, (E)-2-methyl-4-(2,6,6-trimethyl-1-cyclohex-1-yl)-1-buten-1-ol formate and is from the freshwater snail Latia neritoides. Bacterial luciferin is two-component system consisting of flavin mononucleotide and a fatty aldehyde found in bioluminescent bacteria. Coelenterazine Coelenterazine is found in radiolarians, ctenophores, cnidarians, squid, brittle stars, copepods, chaetognaths, fish, and shrimp. It is the prosthetic group in the protein aequorin responsible for the blue light emission.

Official source

https://en.wikipedia.org/wiki/Luciferin

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Bioluminescent imaging is a powerful technique that enables imaging in living organisms with high sensitivity, low background signal, low cost and without the need for radioactivity. The emitted photons are produced in the oxidation reaction of luciferin catalyzed by luciferase. In the caged luciferin approach, the bioluminescent readout is used for measuring the activity of a specific biochemical process. Luciferin is caged in order to prevent catalysis by luciferase, and only when the cage is removed by a specific biochemical event of interest, luciferin is released and becomes a substrate of luciferase. Hence, emitted photons are proportional to the amount of free luciferin, thus allowing to monitor the dynamics of the investigated process. Recent progress in the development of bioorthogonal reactions enabled to answer several fundamental biological questions, improve clinical diagnosis, and to better evaluate therapy efficacy. For example, one of the most robust biorthogonal reactions is the Staudinger ligation, which occurs between a properly tuned azide and a phosphine. In this work, we have used the caged luciferin approach combined with the Staudinger ligation to monitor metabolite uptake in living animals. We have successfully applied this method for the monitoring uptake of two metabolites â glucose and nicotinamide riboside (NR). Glucose is a major source of energy for most living organisms and its aberrant uptake is linked to many pathological conditions. On the other hand, NR is a form of vitamin B3 and represents one of the salvageable NAD+ precursors. Low NAD+ level inside of cells is strongly linked to numerous metabolic and age-related disorders. Despite of their importance, in both cases our understanding of disease-associated glucose or NR flux is limited due to a lack of robust tools. To date, positron emission tomography (PET) imaging remains the gold standard for measuring glucose uptake and no optical tools exist for non-invasive longitudinal imaging of neither of the two metabolites in in vivo settings. Here we report the development of a novel bioluminescent glucose uptake probe (BiGluc) and bioluminescent NR uptake probe (BiNR) for real-time, non-invasive longitudinal imaging of glucose and NR absorption both in vitro and in vivo. The new imaging reagents enable a wide range of applications in the field of metabolism and drug development.

EPFL2020

The Split Luciferin Reaction

Aurélien Godinat

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 Î²-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.

EPFL2017

Caged luciferin probes for the bioluminescence imaging of nitroreductase and hydrogen sulfide in living animals

Anzhelika Vorobyeva

Molecular imaging advances our understanding of cellular and molecular functions within complex biological systems. The development of novel imaging probes that can be applied to answer fundamental biological questions, diagnose and monitor disease progression and the efficacy of therapy in vivo is essential to the field of molecular imaging. Bioluminescence imaging is a powerful technique that allows to study and follow biological processes of interest noninvasively in real-time in living animals. Activatable bioluminescent probes can be designed according to the target of interest and can provide imaging signal in response to the specific stimuli. A strategy called "caged luciferin" can be used to design new bioluminescent probes for imaging of enzymatic activity, the presence of small molecules or metabolite uptake in vivo. In Chapter 1 we present an overview of bioluminescence imaging, with a focus on firefly luciferase-luciferin system. The caged luciferin approach is discussed and examples of existing caged luciferin probes are presented. In Chapter 2 we describe the development of a bioluminescent probe for imaging of bacterial nitroreductase activity and show the potential for its application in different areas of research. Bacterial nitroreductases have been widely utilized in the gene-directed enzyme prodrug therapy (GDEPT) approach for cancer therapy that reached clinical trials. However, both preclinical and clinical development of NTR-based GDEPT systems has been hampered by the lack of imaging tools that allow in vivo evaluation of transgene expression. We developed the NTR caged luciferin (NCL) probe and demonstrated its application for sensitive bioluminescence 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. We anticipate that this probe would significantly accelerate the development of cancer therapy approaches based on GDEPT and other fields where evaluation of NTR expression is important. In Chapter 3 we describe the development of a bioluminescent probe for imaging of hydrogen sulfide (H2S) using the caged luciferin approach. The objective of the research was to develop a probe that can detect endogenously produced H2S noninvasively in real-time in living animals. We present a series of caged luciferin probes that were evaluated on their ability to rapidly and selectively detect H2S in in vitro assays, in cells and in bacterial culture. The two selected probes were applied for imaging of exogenous H2S in the gastrointestinal tract in living mice. We anticipate further applications of the best performing probe (Az-CL) for imaging of H2S production by gut microbiota in mice. In Chapter 4 we present the overall conclusion followed by the outlook and future perspectives.

EPFL2016