Apoptosis is an important and necessary cell death program which promotes homeostasis and organismal survival. When dysregulated, however, it can lead to a myriad of pathologies from neurodegenerative diseases to cancer. Apoptosis is therefore the subject of intense study aimed at dissecting its pathways and molecular mechanisms. Although many assay methods exist for confirming whether an apoptotic response has occurred in vitro, most methods are destructive and involve laborious operator effort or specialized instrumentation. Here we describe a real-time, no-wash, microplate method which utilizes recombinant annexin V fusion proteins containing evolved binary subunits of NanoBiT™ luciferase. The fusion proteins, a time-released enzymatic substrate, a necrosis detection dye and exogenous calcium ions are delivered via an optimized and physiologically inert reagent directly to cells in culture at the time of treatment or dosing. Luminescent signals proportional to phosphatidylserine (PS) exposure and fluorescent signals generated as a result of loss of membrane integrity are then collected using a standard multimode plate reader at scheduled intervals over the exposure. The resulting luminescent and fluorescent data are then used to define the kinetics and magnitude of an apoptotic response. This study details our efforts to develop, characterize, and demonstrate the features of the assay by providing relevant examples from diverse cell models for programmed cell death.

Abstract Efficacious and durable anti-cancer responses are driven by both the selective death of malignant cells and the induction of immunostimulatory activities. Limited but promising clinical evidence suggests that provocation of an unknown balance of inflammatory- and non-inflammatory cell death may be key for orchestrating these positive outcomes. Therefore, identifying and characterizing new clinically useful small molecules and biologic inducers (or combinations thereof) which promote a spectrum of programmed cell death in in vitro screening environments remains critically important. Unfortunately, current screening methods are either insufficiently robust, cost- or resource-prohibitive, or provide no means for initial characterization of the kinetics of programmed cell death. To address this unmet need, we developed a real-time, live cell assay method that utilizes a fully homogeneous, bioluminescent annexin V reagent. The method does not require laborious washing and sample preparation steps associated with traditional annexin methods and is fully compatible with plate-based multimodal signal detection systems. The system contains two annexin proteins which have been engineered to contain separate and distinct complementing domains of a binary luciferase. Additionally, the system contains a novel time-released luciferase substrate and a cell impermeable, fluorogenic DNA dye for monitoring necrosis. Because the annexin-luciferase fusion pairs have only modest affinity for each other, luminescence remains low until phosphatidylserine exposure, a hallmark of the programmed cell death phenotype, brings annexin monomers into close proximity facilitating complementation of the luciferase sensor. The assay reagent can be applied at dosing for real-time measurement of the dose-dependency and magnitude of programmed cell death progression. This work describes our efforts to characterize and validate the performance of the bioluminescent annexin assay using relevant cell death induction models. First, the assay was shown to be functionally concordant with a flow cytometry annexin method in a dose-response model with bortezomib at exposure periods known to produce both early and late-apoptosis (with necrosis) phenotypes. Next, we assessed the performance of the assay using a limited training set of small molecule and biologic inducers of programmed cell death that utilize different mechanisms of action (i.e., apoptosis and necroptosis) and by using a number of diverse but representative cancer cell lines. We conclude that the bioluminescent annexin method provides a new kinetic approach to efficient and effective detection of programmed cell death mechanisms in real time in a convenient homogeneous format. Citation Format: Kevin Kupcho, Andrew Niles, John Shultz, Jamison Grailer, Wenhui Zhou, Robin Hurst, Jim Hartnett, Terry Riss, Dan Lazar, James Cali. A real-time annexin V method for monitoring programmed cell death [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4296. doi:10.1158/1538-7445.AM2017-4296

Abstract The selective elimination of malignant cells via the apoptotic process continues to be the cornerstone of modern anti-cancer therapy regimens. Therefore, in vitro screening approaches aimed at identifying clinically useful apoptosis inducers remain critically important. Recently, phenotypic screening methods have enjoyed a resurgence due to more biologically complex and relevant cell models as well as advances in chemical proteomics which have allowed for more successful target identification. As a consequence, novel probes and tools with enabling attributes are required to fully realize this discovery potential. In an effort to address this unmet need, we have developed a bioluminescent and homogeneous annexin V binding assay for the assessment of apoptosis. Unlike traditional fluorescent annexin V methodology, the “no-wash” reagent employed in this new assay utilizes binary components of a novel luciferase separately fused to annexin V. The annexin V-luciferase subunit fusion pairs have low intrinsic affinity for each other and thus produce no or low luminescence until phosphatidylserine (PtdSer) exposure drives annexin-fusion pair oligimerization. Ultimately, this protein:protein interaction on or near the cell surface reconstitutes full luciferase activity causing an increase in luminescence in the presence of a luciferase substrate. A separate, pro-fluorescent, multiplexed component of the reagent further delineates differences in annexin positivity based on maintenance or loss of membrane integrity corresponding to apoptosis or necrosis, respectively. We validated this method using a panel of diverse cancer cell lines (U2-OS, DLD-1, HeLa, Jurkat, K562, A549, and PC-3), representing both attachment-dependent and -independent morphologies after dose-dependent challenge with intrinsic (bortezomib, panobinostat, staurosporine, and paclitaxel) and extrinsic (rhTRAIL) inducers of apoptosis as well as agents known to produce primary necrosis (ionomycin and digitonin). Caspase activation data was also collected in parallel plates at endpoint as a well-validated and sensitive orthogonal comparator. The bioluminescent annexin V method proved sufficiently robust in 384 well microplate formats to routinely produce Z’ > 0.7 and rank-order potencies in good agreement with caspase activation values. In addition to this microplate functionality, the reagent allowed for sensitive, facile imaging of apoptotic induction in living cells using different imaging platforms. Taken together, the method and reagent should provide unparalleled flexibility with regard to live cell apoptosis detection in both conventional microplate and high content-like imaging formats and advance the pace of new chemical entity discovery. Citation Format: Kevin Kupcho, John Shultz, Andrew Niles, Wenhui Zhou, Robin Hurst, Jim Hartnett, Thomas Machleidt, Terry Riss, Dan Lazar, Jim Cali. A bioluminescent, homogeneous annexin V microplate-based method for assessment of apoptosis. [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 3505.

Induction of oxidative stress by drugs and other xenobiotics is an important mechanism of cytotoxicity. However, in vitro studies on the relationship between oxidative stress and cytotoxicity in cultured cells is frequently complicated by the fact that cell culture medium components affect reactive oxygen species (ROS) exposures in ways that vary with the mode of ROS production. The objectives of this study were to first determine the mode of ROS induction by certain model compounds when they are applied to cultured cells, and then to determine how ROS induction and cytotoxicity were affected by the ROS-quenching medium component pyruvate. Three compounds, eseroline, benserazide, and pyrogallol induced H2O2 in cell culture media independent of cells. However, another compound, menadione, induced H2O2 in a manner largely dependent on the MDA-MB-231 breast cancer cells used in this study, which is consistent with its known mechanism of inducing ROS through intracellular redox cycling. 1 mM pyruvate, as well as catalase, reduced the H2O2 in culture wells with each ROS inducer tested but it only reduced the cytotoxicity of cell-independent inducers. It reduced the cytotoxicity of benserazide and pyrogallol > 10-fold and of eseroline about 2.5-fold, but had no effect on menadione cytotoxicity. From this data, it was concluded that depending on the mechanism of ROS induction, whether intra-or extracellular, a ROS-quenching medium component such as pyruvate will differentially affect the net ROS-induction and cytotoxicity of a test compound.

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.

Abstract 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

Glutathione S-transferases (GSTs) constitute a family of detoxification enzymes that catalyze the conjugation of glutathione with a variety of hydrophobic compounds, including drugs and their metabolites, to yield water-soluble derivatives that are excreted in urine or bile. Profiling the effect of small molecules on GST activity is an important component in the characterization of drug candidates and compound libraries. Additionally, specific GST isozymes have been implicated in drug resistance, especially in cancer, and thus represent potential targets for intervention. To date, there are no sensitive miniaturized high-throughput assays available for GST activity detection. A series of GST substrates containing a masked luciferin moiety have been described recently, offering the potential for configuring a sensitive screening assay via coupled luciferase reaction and standard luminescence detection. We report on the optimization and miniaturization of this homogeneous method to 1,536-well format using GSTs from 3 different species: mouse isozyme A4-4, human isozymes A1-1, M1-1, and P1-1, and the major GST from the parasitic worm Schistosoma japonicum.