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.

Proluciferin acetals as bioluminogenic substrates for cytochrome P450 activity and probes for CYP3A inhibition.

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.

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.

Luminogenic cytochrome P450 assays.

Luminogenic cytochrome P450 (CYP) assays couple CYP enzyme activity to firefly luciferase luminescence in a technology called P450-Glo(TM) (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.

Bioluminescent cell-based NAD(P)/NAD(P)H assays for rapid dinucleotide measurement and inhibitor screening.

Abstract The central role of nicotinamide adenine dinucleotides in cellular energy metabolism and signaling makes them important nodes that link the metabolic state of cells with energy homeostasis and gene regulation. In this study, we describe the implementation of cell-based bioluminescence assays for rapid and sensitive measurement of those important redox cofactors. We show that the sensitivity of the assays (limit of detection ∼0.5 nM) enables the selective detection of total amounts of nonphosphorylated or phosphorylated dinucleotides directly in cell lysates. The total amount of NAD+NADH or NADP+NADPH levels can be detected in as low as 300 or 600 cells/well, respectively. The signal remains linear up to 5,000 cells/well with the maximum signal-to-background ratios ranging from 100 to 200 for NAD+NADH and from 50 to 100 for NADP+NADPH detection. The assays are robust (Z' value >0.7) and the inhibitor response curves generated using a known NAD biosynthetic pathway inhibitor FK866 correlate well with the reported data. More importantly, by multiplexing the dinucleotide detection assays with a fluorescent nonmetabolic cell viability assay, we show that dinucleotide levels can be decreased dramatically (>80%) by FK866 treatment before changes in cell viability are detected. The utility of the assays to identify modulators of intracellular nicotinamide adenine dinucleotide levels was further confirmed using an oncology active compound library, where novel dinucleotide regulating compounds were identified. For example, the histone deacetylase inhibitor entinostat was a potent inhibitor of cellular nicotinamide adenine dinucleotides, whereas the selective estrogen receptor modulator raloxifene unexpectedly caused a twofold increase in cellular nicotinamide adenine dinucleotide levels.

Automated luminescence-based cytochrome P450 profiling using a simple, elegant robotic platform.

The determination of inhibitory effects that lead compounds have on cytochrome P450 (CYP) enzymes is an important part of today's drug discovery process. Assays can be performed early in the discovery process to predict adverse drug-drug interactions caused by CYP inhibition and to minimize the costs associated with terminating candidates in late stage development or worse, removing a drug from the market after launch. For early discovery work, testing substantial numbers of compounds is desirable, thus automated "mix and read" assays are beneficial. Here, we demonstrate the automation of the CYP profiling process using a simple, yet robust robotic platform. Compound titration, as well as transfer of compounds and assay components was performed by the same automated pipetting system. IC(50)s of small molecule drugs were determined using recombinant CYP enzymes, CYP3A4, -2C9, and -2D6 and luminogenic substrates specific to each. Compounds were profiled against all three enzymes on the same 384-well assay plate.

The utility of semi-automating multiplexed assays for ADME/Tox applications.

ADME-Tox testing examines the effects of an organism, tissue or cell on a compound, as well as the effects that the compound has on an organism, tissue or cell, and has gained in importance in the overall drug discovery process over the past twenty years. This is due to the rising percentage of drug candidate attrition in the 1990s and early 2000s due to adverse ADME/Tox profiles. The increased importance placed upon ADME/Tox testing has brought about new types of in-vitro assay technologies utilizing microplates to deliver the most pharmacologically relevant data. These tests, however, have typically been performed sequentially, where multiple assays over multiple microplates are used. This typically leads to increased time and cost required to generate the required information, and can sacrifice data quality. Multiplexed assays, however, where more than one piece of data can be attained from a single well or a single microplate, performed using appropriate liquid handling and detection instrumentation, can improve data quality and reduce the time and expense required to attain this information.