Search

Bioluminescence methodologies have been extraordinarily useful due to their high sensitivity, broad dynamic range, and operational simplicity. These capabilities have been realized largely through incremental adaptations of native enzymes and substrates, originating from luminous organisms of diverse evolutionary lineages. We engineered both an enzyme and substrate in combination to create a novel bioluminescence system capable of more efficient light emission with superior biochemical and physical characteristics. Using a small luciferase subunit (19 kDa) from the deep sea shrimp Oplophorus gracilirostris, we have improved luminescence expression in mammalian cells ∼2.5 million-fold by merging optimization of protein structure with development of a novel imidazopyrazinone substrate (furimazine). The new luciferase, NanoLuc, produces glow-type luminescence (signal half-life >2 h) with a specific activity ∼150-fold greater than that of either firefly (Photinus pyralis) or Renilla luciferases similarly configured for glow-type assays. In mammalian cells, NanoLuc shows no evidence of post-translational modifications or subcellular partitioning. The enzyme exhibits high physical stability, retaining activity with incubation up to 55 °C or in culture medium for >15 h at 37 °C. As a genetic reporter, NanoLuc may be configured for high sensitivity or for response dynamics by appending a degradation sequence to reduce intracellular accumulation. Appending a signal sequence allows NanoLuc to be exported to the culture medium, where reporter expression can be measured without cell lysis. Fusion onto other proteins allows luminescent assays of their metabolism or localization within cells. Reporter quantitation is achievable even at very low expression levels to facilitate more reliable coupling with endogenous cellular processes.

The cytochrome P450s (CYPs) are central to ADME studies because of their central role in drug metabolism. Proper CYP assay design and a correct understanding of CYP assay selectivity are critical for generating and interpreting biologically relevant data during drug development. Bioluminescent CYP assays use luminogenic probe substrates that have the unique property of producing photons in a second reaction with luciferase.This article presents the general design principles for in vitro CYP assays. Specifically, the article focuses on the bioluminescent approach that couples CYP activity with photon production.Highly selective luminogenic substrates for CYP1A1, CYP1A2, CYP2C9, CYP3A4, CYP3A7, CYP4A and CYP4F have been developed with utility for interrogating the roles of these enzymes in biochemical and cell-based formats. These selective substrates are part of a larger collection of probes that deliver CYP inhibition and induction data that predict in vivo drug interactions. Furthermore, they support highly sensitive, rapid and scalable assays for cell-based and cell-free biochemical applications, which offer an alternative and often enabling option over conventional assay strategies.

Five novel firefly luciferin analogues in which the benzothiazole ring system of the natural substrate was replaced with benzimidazole, benzofuran, benzothiophene, benzoxazole, and indole were synthesized. The fluorescence, bioluminescence, and kinetic properties of the compounds were evaluated with recombinant Photinus pyralis wild type luciferase. With the exception of indole, all of the substrates containing heterocycle substitutions produced readily measurable flashes of light with luciferase. Compared to that of luciferin, the intensities ranged from 0.3 to 4.4% in reactions with varying pH optima and times to reach maximal intensity. The heteroatom changes influenced both the fluorescence and bioluminescence emission spectra, which displayed maxima of 479-528 and 518-574 nm, respectively. While there were some interesting trends in the spectroscopic and bioluminescence properties of this group of structurally similar substrate analogues, the most significant findings were associated with the benzothiophene-containing compound. This synthetic substrate produced slow decay glow kinetics that increased the total light-based specific activity of luciferase more than 4-fold versus the luciferin value. Moreover, over the pH range of 6.2-9.4, the emission maximum is 523 nm, an unusual 37 nm blue shift compared to that of the natural substrate. The extraordinary bioluminescence properties of the benzothiophene luciferin should translate into greater sensitivity for analyte detection in a wide variety of luciferase-based applications.

The aldehyde dehydrogenase (ALDH) family of enzymes is critical for cell survival and adaptation to cellular and environmental stress. These enzymes are of interest as therapeutic targets and as biomarkers of stem cells. This article describes a novel, homogeneous bioluminescence assay to study the activity of the ALDH enzymes. The assay is based on a proluciferin–aldehyde substrate that is recognized and utilized by multiple ALDH enzyme isoforms to generate luciferin. A detection reagent is added to inactivate ALDH and generate light from the luciferin product. The luminescent signal is dependent on the ALDH enzyme concentration and the incubation time in the ALDH reaction; moreover, the luminescent signal generated with the detection reagent is stable for greater than 2 h. This assay provides many advantages over standard NADH fluorescence assays. It is more sensitive and the signal stability provided allows convenient assay setup in batch mode-based high-throughput screens. The assay also shows an accurate pharmacological response for a common ALDH inhibitor and is robust, with a large assay window (S/B = 64) and Z′ = 0.75.

Cytochrome P450 (P450) assays use probe substrates to interrogate the influence of new chemical entities toward P450 enzymes. We report the synthesis and study of a family of bioluminogenic luciferin acetal substrates that are oxidized by P450 enzymes to form luciferase substrates. The luciferin acetals were screened against a panel of purified P450 enzymes. In particular, one proluciferin acetal has demonstrated sensitive and selective CYP3A4-catalyzed oxidation to a luciferin ester-K(m) and k(cat) are 2.88 μM and 5.87 pmol metabolite · min(-1) · pmol enzyme(-1), respectively. The proluciferin acetal was used as a probe substrate to measure IC(50) values of known inhibitors against recombinant CYP3A4 or human liver microsomes. IC(50) values for the known inhibitors correlate strongly with IC(50) values calculated from the traditional high-performance liquid chromatography-based probe substrate testosterone. Luciferin acetals are rapidly oxidized to unstable hemi-orthoesters by CYP3A resulting in luciferin esters and, therefore, are conducive to simple rapid CYP3A bioluminescent assays.

A set of 6'-alkylated aminoluciferins are shown to be bioluminescent substrates for Ultra-Glo and QuantiLum luciferases. These studies demonstrate that both the engineered and wild-type firefly luciferases tolerate much greater steric bulk at the 6' position of luciferin than has been previously reported. The nature of the alkyl substituent strongly affects the strength of the bioluminescent signal, which varies widely based on size, shape, and charge. Several compounds were observed to generate more light than the corresponding unsubstituted 6'-aminoluciferin. Determination of Michaelis-Menten constants for the substrates with Ultra-Glo indicated that the variation arises primarily from differences in V max, ranging from 1.33 x 10 (4) to 332 x 10 (4) relative light units, but in some cases K m (0.73-10.8 microM) also plays a role. Molecular modeling results suggest that interactions of the side chain with a hydrogen-bonding network at the base of the luciferin binding pocket may influence substrate-enzyme binding.

Cell-based assay development for cancer drug discovery continues to advance toward more innovative methods that provide efficient workflow and meaningful data. Rapid, sensitive techniques that are non-lytic and therefore facilitate multiplexing with a wide range of assays allow more data to be gained per well and conserve precious samples. Real time measurement of cell viability also allows numerous measurements over time without the need for separate plates at each time point. Real time measurements allow unique analyses including interrogation of the timing of drug killing, identification of static vs toxic drug effects, and determination of the ideal time to multiplex an additional assay. Cancer drug discovery would benefit from novel assay technologies that allow real time measurements with multiplexing capability. We have developed a homogeneous, non-lytic, and bioluminescent method to analyze cell viability in real time through measurement of the reducing potential of the cell. The signal correlates with the number of viable cells making it well-suited for cytotoxicity studies. This Real Time Cell Viability Assay utilizes the NanoLuc luciferase enzyme, which is 100-fold brighter than either firefly (Photinus pyralis) or Renilla reniformis luciferase, and the novel substrate, furimazine. Together these reagents produce high intensity luminescence through an ATP-independent reaction. The assay can be performed in two formats, live cell endpoint or continuous read. The live cell endpoint method is sensitive, rapid, and enables extensive multiplexing opportunities due to the non-lytic measurement and signal depletion upon cell lysis. When the assay is set up in the continuous read format, the cell viability reagents can be added at the same time as cell plating, drug dosing, or at whatever point in the assay the researcher would like to start obtaining viability readings. The luminescent signal can be continually monitored from the same wells over an extended period of time to analyze cell viability in real time. This real time cell viability assay enables non-lytic cell viability measurements, extensive multiplexing options including both fluorescent and luminescent assays with no special filter requirements, straightforward normalization studies, conservation of precious samples and reagents, and a sensitive, real time measurement of cell viability. Citation Format: Sarah J. Duellman, Jolanta Vidugiriene, Wenhui Zhou, Jean Osterman, Ruslan Arbit, Laurent Bernad, Poncho Meisenheimer, James J. Cali. Bioluminescent, non-lytic, real-time cell viability assay. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3743. doi:10.1158/1538-7445.AM2014-3743

Cell metabolism is an important and expanding area of research in the field of cancer biology. Simple, rapid assays for studying cellular metabolic pathways, their enzymes, metabolites and cofactors can facilitate these studies. We have developed two novel bioluminescent technologies, one for monitoring cell viability, and the other for monitoring changes in cellular nicotinamide adenine dinucleotides, that can facilitate the study of cell metabolism and its regulation. The bioluminescent cell viability assay is based on the metabolic activity of live cells. Metabolically active cells reduce a proluminogenic substrate in vivo which is then detected by a luciferase enzyme in the media. The luminescence output is correlated with the number of viable cells. The assay is non-toxic, sensitive (detects less than 10 cells/well) and has a large dynamic range (S/B>100). The key, differentiating feature of this assay is the incorporation of the recently developed stable and very bright NanoLuc™ luciferase and its profurimazine substrate, which allows changes in viability to be monitored in real-time and continuously, over extended periods of time, from the same sample. Three homogeneous, one-step, bioluminescent assays have been developed for the detection of nicotinamide adenine dinucleotides, specific for: (1) reduced forms NADH and NADPH, (2) non-phosphorylated forms NAD and NADH, and (3) phosphorylated forms NADP and NADPH. The assays are based on the reaction of the enzyme diaphorase with a proluciferin substrate, which in the presence of NADH or NADPH is converted to luciferin, a substrate for luciferase. These assays are sensitive (LOD ≤ 50 nM) and have large maximum signal windows (S/B > 100), two advantages over currently available colorimetric and fluorescent detection methods. They are also robust, and amenable to automated and high-throughput protocols (Z’ > 0.8). The high sensitivity, one-step reagent addition, and no requirement for sample processing, permit an in-well protocol for the assay of cells directly in culture plate wells. Changes in cellular NAD levels can be rapidly monitored, as demonstrated using FK866, an inhibitor of NAD biosynthesis. FK866 caused a decrease in NAD levels in multiple cell types with good signal windows (S/B > 10) and expected pharmacology (IC50 values = 1-10 nM). Citation Format: Donna Leippe, Mary Sobol, Sarah Duellman, Jolanta Vidugiriene, Wenhui Zhou, Gediminas Vidugiris, Troy Good, Laurent Bernad, Poncho Meisenheimer, James Cali. Bioluminescent assays for investigating cell metabolism. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1896. doi:10.1158/1538-7445.AM2013-1896

Cancer is a disease defined by uncontrolled cell growth where cellular energy metabolism pathways must evolve for tumors to survive and proliferate. NAD(P) and NAD(P)H play a major role in oxidative phosphorylation and their role in aerobic glycolysis is of great interest. Additionally, NAD(P)/NAD(P)H act as co-factors for enzymes involved in cancer pathogenesis through regulation of chromatin structure, DNA repair, and transcription (e.g. sirtuins and poly(ADP-ribose) polymerase). The study of how NAD(P)/NAD(P)H are generated and utilized during adaptive cancer cell energy metabolism would benefit from the development of a rapid, sensitive, and homogeneous assay to measure the level of these nucleotides. We developed a bioluminescent assay for measuring NAD(P)/NAD(P)H that can detect ≤ 0.1 µM NADH and has a 1000-fold dynamic range. The assay is well suited for high throughput screening (Z’ = 0.92, S/B = 95) and has been used to screen the LOPAC library. By coupling other reactions with NAD(P)/NAD(P)H measurement, we analyzed the levels of metabolites and the activity of enzymes, including isocitrate dehydrogenase and pyruvate dehydrogenase, in enzyme preparations and crude cell lysates. This method is applicable for measuring cellular NAD and NADH levels directly from cell culture without further sample handling. This assay is based on a novel proluciferin derivative that is processed in vitro through an enzymatic reaction. The released luciferin is detected in a coupled luciferin/luciferase reaction and the luminescent signal is correlated with the amount of NAD(P)/NAD(P)H present in the sample. As an extension of this approach, similar proluciferin chemistries were developed to detect the reducing potential of metabolically active cells. This robust luminescent assay can detect the reducing potential of less than 100 cells/well in 96-well format and distinguish small changes in cell number. Multiple cell lines were treated with cancer therapeutic compounds and the performance of the proluciferin approach was compared to commonly used colorimetric (e.g. MTT, MTS, and XTT) and fluorogenic (e.g. resazurin) methods. The proluciferin assay resulted in comparable pharmacological responses, significantly increased signal window, and superior sensitivity. Additionally, the proluciferin-based approach has been extended to other metabolic readouts. For example, novel proluciferin substrates were developed and applied to the detection of hydrogen peroxide in cells and in biological samples through a HRP-independent process. Bioluminescent assays provide greater sensitivity and dynamic range than most fluorescent or colorimetric assays, and are better suited for high throughput screening. Applying these assays to the study of cancer cell energy metabolism will provide a significant advantage over existing methods. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1135. doi:1538-7445.AM2012-1135