Donna Leippe

Self-Immolative Bioluminogenic Quinone Luciferins for NAD(P)H Assays and Reducing Capacity-Based Cell Viability Assays

Highly sensitive self‐cleavable trimethyl lock quinone‐luciferin substrates for diaphorase were designed and synthesized to measure NAD(P)H in biological samples and monitor viable cells via NAD(P)H‐dependent cellular oxidoreductase enzymes and their NAD(P)H cofactors.

A bioluminescent assay for the sensitive detection of proteases.

A bioluminescent general protease assay was developed using a combination of five luminogenic peptide substrates. The peptide-conjugated luciferin substrates were combined with luciferase to form a homogeneous, coupled-enzyme assay. This single-reagent format minimized backgrounds, gave stable signals, and reached peak sensitivity within 30 min. The bioluminescent assay was used to detect multiple proteases representing serine, cysteine, and metalloproteinase classes. The range of proteases detected was broader and the sensitivity greater, when compared with a standard fluorescent assay based on cleavage of the whole protein substrate casein. Fifteen of twenty proteases tested had signal-to-background ratios >10 with the bioluminescent method, compared with only seven proteases with the fluorescent approach. The bioluminescent assay also achieved lower detection limits (≤100 pg) than fluorescent methods. During protein purification processes, especially for therapeutic proteins, even trace levels of contamination can impact the proteins stability and activity. This sensitive, bioluminescent, protease assay should be useful for applications in which contaminating proteases are detrimental and protein purity is essential.

Bioluminescent Assays for Glucose and Glutamine Metabolism High-Throughput Screening for Changes in Extracellular and Intracellular Metabolites

Cancer cell metabolism is a complex, dynamic network of regulated pathways. Interrogation of this network would benefit from rapid, sensitive techniques that are adaptable to high-throughput formats, facilitating novel compound screening. This requires assays that have minimal sample preparation and are adaptable to lower-volume 384-well formats and automation. Here we describe bioluminescent glucose, lactate, glutamine, and glutamate detection assays that are well suited for high-throughput analysis of two major metabolic pathways in cancer cells: glycolysis and glutaminolysis. The sensitivity (1–5 pmol/sample), broad linear range (0.1–100 µM), and wide dynamic range (>100-fold) are advantageous for measuring both extracellular and intracellular metabolites. Importantly, the assays incorporate rapid inactivation of endogenous enzymes, eliminating deproteinization steps required by other methods. Using ovarian cancer cell lines as a model system, the assays were used to monitor changes in glucose and glutamine consumption and lactate and glutamate secretion over time. Homogeneous formats of the lactate and glutamate assays were robust (Z′ = 0.6–0.9) and could be multiplexed with a real-time viability assay to generate internally controlled data. Screening a small-compound library with these assays resulted in the identification of both inhibitors and activators of lactate and glutamate production.

Abstract B49: Bioluminescent assays for measuring glycolytic rate and glucose homeostasis

We describe a set of bioluminescence assays for systematic evaluation of metabolic alterations in response to different growth conditions or treatments. These newly developed cell-based bioluminescence assays offer new methods to detect different metabolites such as NAD(P)/NAD(P)H, lactate, glutamate, glucose-6-phosphate, glucose and 2-deoxyglucose-6-phosphate (for glucose uptake). These assays address the need for more rapid, sensitive and easier to use approaches to quantify key metabolites in cell lysates and tissues. Using the glucose uptake assay, compounds altering translocation of glucose transporters can be screened and characterized rapidly. The lactate detection assay offers a sensitive and rapid approach for glycolytic rate measurements. The glucose detection assay provides information on glucose consumption rates. When these glucose consumption rates are correlated with lactate secretion, the results serve as an indicator of the metabolic status of the cells (e.g. a shift from oxidative phosphorylation to glycolysis). The glucose assay also provides a convenient and robust approach for studying activation or inhibition of gluconeogenesis or glycogenolysis. These bioluminescent assays are robust and sensitive with assay windows significantly larger than analogous fluorescent or colorimetric methods. The sensitivity (1-5pmol/sample) and wide dynamic range (maximum signal-to-background > 100 fold) of the assays allow simultaneous detection of multiple metabolites from the same set of samples. Most importantly, the assays incorporate rapid inactivation of endogenous enzymes, which overcomes the limitations of the deprotonization step required by other methods. The improved workflow in combination with the high sensitivity of luminescence enables measurement of intracellular metabolite levels by simply adding detection reagents directly to treated cells. Alternatively, small samples (2-5ul) can be removed from the media and used for multiple metabolite (lactate, glucose, glutamate) measurements. By consolidating dose- and time-dependent measurements, these assays conserve cells and reagents, streamline workflow, and produce internally controlled data sets. The assays can be used in various formats (e.g. 96- and 384-well plates), applied to various sample types (e.g. mammalian cells, tissues, and 3D microtissues), and multiplexed with other assays, including our new real time cell viability assay. The bioluminescent real time cell viability assay is a homogeneous, non-lytic method to measure cell viability in real time. It overcomes the limitations of standard end-point lytic assays by providing a comprehensive representation of changes in cell viability occurring in live cells over the entire length of an experiment. Changes in cell viability can be conveniently correlated with changes in metabolite levels. Citation Format: Jolanta Vidugiriene, Donna Leippe, Mary Sobol, Mike Valley, Natasha Karassina, Sarah Duellman, James Cali. Bioluminescent assays for measuring glycolytic rate and glucose homeostasis. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr B49.

Abstract 1057: Bioluminescent assays for investigating cellular energy metabolism

Cellular energy metabolism is recognized to have a central role in many cell processes and is an increasingly important area of study in many research fields such as cancer, immunology, obesity, diabetes and neurology. To facilitate these studies, we have taken advantage of key features of bioluminescence, such as increased sensitivity and wide assay windows, to develop assays for measuring energy metabolites. These assays are easy-to-use plate-based assays with streamlined protocols that require minimal cell sample processing. The assays can be used to measure consumption of major fuel sources such as glucose and glutamine from the cell medium, as well as lactate and glutamate secretion. The sensitivity (1-5pmol/sample), broad linear range (0.1-100 uM) and wide dynamic range (maximum signal-to-background > 100 fold) allow all four metabolites to be measured from the same sample of medium and over time, with earlier detection from fewer cells compared to other methods. The assays are also sensitive enough to detect intracellular levels of glutamine, glutamate and lactate and changes in those levels. Additional assays have been developed to measure glucose uptake and NAD/NADH levels in cells. Analysis of these metabolites provides information regarding the energetic state of the cell. In this poster we provide examples demonstrating the utility of these assays to detect a glycolytic shift in cancer cells, monitor cell growth and differentiation with associated changes in glucose uptake and glycolysis, and evaluate insulin sensitivity in primary adipocytes. Citation Format: Donna M. Leippe, Mary Sobol, Mike Valley, Natasha Karassina, Sarah Duellman, Jolanta Vidugiriene, Jim Cali. Bioluminescent assays for investigating cellular energy metabolism. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1057.

Abstract 1211: Metabolite assays to illuminate cellular energy networks

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Every organism requires cellular energy metabolism to meet developmental needs. When cells change from a resting to a proliferative state, the metabolic pathways are reprogrammed to meet the demands of proliferation. Diseases such as cancer can occur when changes in the metabolic pathways support unwanted functions such as uncontrolled proliferation. Assays for analyzing metabolites and metabolic enzymes are needed to understand cellular metabolic requirements and energy network regulation under normal and disease conditions. We exploited the robustness, sensitivity and broad dynamic range of bioluminescence detection to develop assays for the key metabolites lactate, glucose, glutamate and glucose-6-phosphate. The technology involves using specific dehydrogenases to couple metabolite levels to NAD(P)H production. In the presence of NAD(P)H, a novel proluciferin is converted to a substrate for luciferase resulting in light signals proportional to the starting metabolite concentration. Samples (e.g. mammalian cells, culture media, tissues, 3D microtissues) are added to the metabolite detection reagent in a 1:1 ratio. An inactivation solution added to complex biological samples lowers the background and increases sensitivity by lysing the cells, inactivating cellular enzymes, and destroying endogenous NAD(P)H. The sensitivity (1-5 pmol/sample) and broad assay window (maximum signal-to-background > 100 fold) of the assays allows simultaneous detection of multiple metabolites from the same set of samples. The wide assay window allows detection of very small changes in metabolite concentration, an advantage over conventional fluorescent and colorimetric detection methods. The assays adapt to various formats (96, 384-well plates) and require no instrumentation other than a luminometer. The assays can be used to measure the levels of critical metabolites and to monitor the effects of inhibitors and activators of glycolysis. The shift from oxidative phosphorylation to glycolysis can be monitored by measuring lactate production and secretion as well as glucose consumption from the same set of samples. The assays easily multiplex with cell viability assays enabling analysis of drug effects on cell proliferation, cell metabolism and mitochondria function. Citation Format: Mary Sobol, Jolanta Vidugiriene, Donna Leippe, Gediminas Vidugiris, Wenhui Zhou, James Cali. Metabolite assays to illuminate cellular energy networks. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1211. doi:10.1158/1538-7445.AM2015-1211

Abstract 1429: Bioluminescent methods for investigating metabolic pathways

Cancer cells are dependent on metabolic pathways that have been altered to support unique requirements for cancer cell growth and survival. The central role of these pathways makes them attractive targets for new treatment approaches. Rapid and sensitive assays, amenable to high-throughput screening, are needed to study the enzymes, metabolites and cofactors involved in cancer cell metabolic pathways. We developed a bioluminescent technology to address these needs and monitor changes in the fundamental metabolic co-factors nicotinamide adenine dinucleotides and key metabolites such as lactate. The bioluminescent method uses a novel proluciferin substrate for the enzyme diaphorase. Luciferin is produced in the presence of NAD(P)H, resulting in a luciferase-generated light signal proportional to the starting NAD(P)H concentration. This is a versatile technology for measuring dinucleotide concentrations, enzyme activity and also metabolite levels. When lactate dehydrogenase is included in the reaction, the cellular metabolite lactate, an indicator of glycolytic rate, can be measured. NAD, NADP, NADH and NADPH can be measured in enzyme reactions or in biological samples, such as cells and tissues, with the use of NAD- and NADP-specific cycling enzymes. Total NAD+NADH or NADP+NADPH levels in cultured cells were rapidly monitored using a one-reagent addition, in-well plate protocol without the need for cell processing. Additionally, the levels of individual dinucleotides and their ratios were determined from cell and tissue samples using a streamlined in-well acid and base-treatment protocol. The effects of small molecules on cellular dinucleotide levels can be quickly assessed, as demonstrated with FK866, an inhibitor of NAD biosynthesis. Advantages of the bioluminescent approach over other detection methods include higher sensitivity (LOD ≤ 50 nM), larger maximum signal windows (S/B > 100) and compatibility with automated, high-throughput protocols (Z9 > 0.8). By providing these features, the bioluminescent assays should be useful tools for facilitating the study of cancer cell metabolic pathways and the development of therapeutics that target these pathways. Citation Format: Donna Leippe, Mary Sobol, Jolanta Vidugiriene, Wenhui Zhou, Gediminas Vidugiris, Troy Good, Laurent Bernad, Poncho Meisenheimer, James Cali. Bioluminescent methods for investigating metabolic pathways. [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 1429. doi:10.1158/1538-7445.AM2014-1429

Abstract 1896: Bioluminescent assays for investigating cell metabolism.

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

Abstract 1135: Bioluminescent methods to investigate cellular metabolic status: NAD(P)/NAD(P)H levels, redox potential, and hydrogen peroxide concentration

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL 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

Reversed enzyme activity DNA interrogation test: a new method for mutation detection

The Reversed Enzyme Activity DNA Interrogation Test (READITTM) System provides a new, rapid method for genotype determination. The system can be used for the analysis of single nucleotide polymorphisms as well as insertions, deletions and chromosomal rearrangements. The READITTM System is based upon driving various enzymatic reactions in the reverse direction from the direction they are normally considered. The method is based on a pyrophosphorylation reaction that is the reverse of DNA polymerization. Interrogation probes annealed to DNA fragments with which they have a 3- mismatch are not a substrate for the reaction and do not generate significant signals compared with probes that match the interrogation sequence perfectly. By interrogation of specific DNA sequences with carefully designed probes, mutant genes can be identified with speed and precision.