Troy Good

Luminogenic cytochrome P450 assays

Luminogenic cytochrome P450 (CYP) assays couple CYP enzyme activity to firefly luciferase luminescence in a technology called P450-GloTM (Promega). Luminogenic substrates are used in assays of human CYP1A1, -1A2, -1B1, -2C8, -2C9, -2C19, -2D6, -2J2, -3A4, -3A7, -4A11, -4F3B, -4F12 and -19. The assays detect dose-dependent CYP inhibition by test compounds against recombinant CYP enzymes or liver microsomes. Induction or inhibition of CYP activities in cultured hepatocytes is measured in a nonlytic approach that leaves cells intact for additional analysis. Luminogenic CYP assays offer advantages of speed and safety over HPLC and radiochemical-based methods. Compared with fluorogenic methods the approach offers advantages of improved sensitivity and decreased interference between optical properties of test compound and CYP substrate. These homogenous assays are sensitive and robust tools for high-throughput CYP screening in early drug discovery.

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.

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