Laurent Bernad

Cell-based bioluminescent assays for all three proteasome activities in a homogeneous format

A luminescent method to individually measure the chymotrypsin-like, trypsin-like, or caspase-like activities of the proteasome in cultured cells was developed. Each assay uses a specific luminogenic peptide substrate in a buffer optimized for cell permeabilization, proteasome activity, and luciferase activity. Luminescence is generated in a coupled-enzyme format in which proteasome cleavage of the peptide conjugated substrate generates aminoluciferin, which is a substrate for luciferase. The homogeneous method eliminates the need to prepare individual cell extracts as samples. Luminogenic proteasome substrates and buffer formulations enabled development of a single reagent addition method with adequate sensitivity for 96- and 384-well plate formats. Proteasome trypsin-like specificity was enhanced by incorporating a mixture of protease inhibitors that significantly reduce nonspecific serum and cellular backgrounds. The assays were used to determine EC(50) values for the specific proteasome inhibitors epoxomicin and bortezomib for each of the catalytic sites using a variety of cancer lines. These cell-based proteasome assays are direct, simple, and sensitive, making them ideal for high-throughput screening.

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.

A bioluminescent caspase-1 activity assay rapidly monitors inflammasome activation in cells ☆

Inflammasomes are protein complexes induced by diverse inflammatory stimuli that activate caspase-1, resulting in the processing and release of cytokines, IL-1β and IL-18, and pyroptosis, an immunogenic form of cell death. To provide a homogeneous method for detecting caspase-1 activity, we developed a bioluminescent, plate-based assay that combines a substrate, Z-WEHD-aminoluciferin, with a thermostable luciferase in an optimized lytic reagent added directly to cultured cells. Assay specificity for caspase-1 is conferred by inclusion of a proteasome inhibitor in the lytic reagent and by use of a caspase-1 inhibitor to confirm activity. This approach enables a specific and rapid determination of caspase-1 activation. Caspase-1 activity is stable in the reagent thereby providing assay convenience and flexibility. Using this assay system, caspase-1 activation has been determined in THP-1 cells following treatment with α-hemolysin, LPS, nigericin, gramicidin, MSU, R848, Pam3CSK4, and flagellin. Caspase-1 activation has also been demonstrated in treated J774A.1 mouse macrophages, bone marrow-derived macrophages (BMDMs) from mice, as well as in human primary monocytes. Caspase-1 activity was not detected in treated BMDMs derived from Casp1-/- mice, further confirming the specificity of the assay. Caspase-1 activity can be measured directly in cultured cells using the lytic reagent, or caspase-1 activity released into medium can be monitored by assay of transferred supernatant. The caspase-1 assay can be multiplexed with other assays to monitor additional parameters from the same cells, such as IL-1β release or cell death. The caspase-1 assay in combination with a sensitive real-time monitor of cell death allows one to accurately establish pyroptosis. This assay system provides a rapid, convenient, and flexible method to specifically and quantitatively monitor caspase-1 activation in cells in a plate-based format. This will allow a more efficient and effective assessment of inflammasome activation as well as enable high-throughput screening for inflammasome modulators.

Abstract 5446: Continuous real-time measurement of live and dead cells in culture over multiple days

Recently developed assay technologies have made it possible to use a multi-well plate reader to estimate the number of live or dead cells in culture in real time over a period of days to monitor the effects of long term exposure to test substances. An advantage of the real time assay chemistries is that the cells remain viable and available for other measurements. The purpose of this study was to determine if the real time assay methods could be multiplexed with other assay endpoints and applied to three dimensional (3D) culture models. The measurement of the number of live cells in real time is accomplished by providing a pro-substrate that viable cells (but not dead cells) can convert into a substrate for a luciferase derived from a marine shrimp. The pro-substrate and luciferase can be added with a single reagent addition step and because they are not toxic, they can be incubated with cells in culture for up to three days. Live cell conversion of the pro-substrate into a substrate for luciferase results in a luminescent signal that is proportional to the number of live cells. The signal diminishes immediately upon cell death. Detection of dead cells in real time is accomplished by using a fluorogenic DNA binding dye that is non-permeant and thus non-toxic to live cells. The dye enters dead cells that have a compromised membrane, binds to DNA, and becomes fluorescent. We have demonstrated that the real time assays can be multiplexed to measure both live and dead cells in the same sample. We further demonstrate the use of these assays to measure viability of cells in 3D spheroids formed using the hanging drop culture technique. We also demonstrate multiplexing of real time assays with subsequent measurement of apoptosis or extraction of RNA for expression studies. Utilization of real time assay chemistries that are non-toxic enabled repeated recording of data in a kinetic mode from the same sample of cells which eliminated the need for duplicate cultures and provided flexibility during assay protocol development. The ability to multiplex real time assays improved overall lab efficiency by eliminating the need for duplicate cultures for each experiment. Citation Format: Terry Riss, Jolanta Vidugiriene, Sarah Duellman, Wenhui Zhou, Gediminas Vidugiris, Mike Valley, Jean Osterman, Ruslan Arbit, Laurent Bernad, Poncho Meisenheimer, James J. Cali. Continuous real-time measurement of live and dead cells in culture over multiple days. [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 5446. doi:10.1158/1538-7445.AM2015-5446

Abstract 1319: Monitoring inflammasome activation with a bioluminescent caspase-1 assay

Inflammatory responses and immune modulation play important and complex roles in cancer development and therapy, but methods to monitor caspase-1 activity associated with inflammasome activation have been limited. Inflammasomes are protein complexes induced by diverse inflammatory stimuli. Caspase-1, an essential component of the inflammasome, is activated in response to these stimuli, resulting in the processing and release of cytokines, IL-1β and IL-18, and pyroptosis, an immunogenic form of cell death. Western blots and ELISA are the primary, but indirect, methods for monitoring caspase-1 activity currently in use. To simplify and provide a more direct means of detecting cell-based caspase-1 activity, we developed a sensitive, homogeneous, plate-based assay that eliminates the need for significant sample processing. The assay employs a single-step, bioluminescent format combining a caspase-1 substrate, Z-WEHD-aminoluciferin, with a thermostable luciferase in an optimized reagent subsequently added to treated cells in an assay well. The coupled-enzyme system quickly reaches a steady-state between caspase cleavage of the substrate and luciferase conversion of the aminoluciferin, with light generated proportional to the amount of caspase-1 activity present in the sample. In addition to substrate selection, assay specificity for caspase-1 is conferred by inclusion of a proteasome inhibitor, MG132, in the reagent and by the subsequent use of a caspase-1 inhibitor, Ac-YVAD-CHO, to confirm activity. This approach enables clear determination of caspase-1 activity even in the context of apoptotic cells. Studies with Casp1 −/− cells further demonstrate the effectiveness of this assay system to specifically detect cell-based, caspase-1 activity. Using this novel assay system, caspase-1 activation has been quantitatively determined in THP-1 cells following treatment with α-hemolysin, LPS, nigericin, monosodium urate crystals, R-848, Pam3CSK4, and flagellin. Caspase-1 activation has also been demonstrated in treated J774A.1 mouse macrophages, bone marrow-derived macrophages from mice, as well as in human primary monocytes. Of note, caspase-1 activity can be monitored either directly in cells or released into the culture medium following cell treatment with various inflammatory stimuli. Monitoring released caspase-1 activity in supernatants is fast, sensitive, and nondestructive, thereby enabling subsequent multiplexing of the biological sample with other assays to monitor additional cell parameters, such as IL-1β release or cell death. Therefore, this assay system provides a rapid, convenient, and flexible method to specifically and quantitatively monitor caspase-1 activation in cells in a plate-based format. This will allow a more efficient and effective assessment of inflammasome activation as well as enable high-throughput screening for inflammasome modulators. Citation Format: Martha O9Brien, Danielle Moehring, Raul Munoz-Planillo, Gabriel Nunez, Justin Callaway, Jenny Ting, Mike Scurria, Tim Ugo, Laurent Bernad, James Cali, Dan Lazar. Monitoring inflammasome activation with a bioluminescent caspase-1 assay. [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 1319. doi:10.1158/1538-7445.AM2015-1319

Abstract 3743: Bioluminescent, non-lytic, real-time cell viability assay

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

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 3885: A bi-functional fluorescent assay for the assessment of cytotoxicity and proliferation

Cell-based models continue to be critical tools for cancer research. Therefore, the assessment of cytotoxic or cytostatic effects remains an obligate experimental activity. Although numerous enzyme activity chemistries exist for measuring cytotoxicity, biomarker degradation may cause underestimation of actual cytotoxicity and limit the utility of these activity measures when ascertained with long-term drug exposures (72hrs or more). We have developed a cell-impermeant probe which labels the DNA from cells with compromised membranes. The quantum efficiency (i.e. brightness) of this dye increases dramatically upon DNA binding, producing a fluorescent signal which can be measured using standard “green” fluorometry wavelengths (excitation 485nm/emission 530nm). Additionally, the dye is non-toxic and stable, allowing for introduction of the probe directly into the culture medium or compound dilutions prior to cell dosing. This “no step” addition feature allows for the measurement of cytotoxicity in real time or at a convenient endpoint. Furthermore, the dye is fully compatible with luminescent measures, allowing for same-well, multi-parametric assessment of the cytotoxic phenotype. Lastly, proliferative or anti-proliferative effects (relative to control) can be revealed by the introduction of a lytic detergent which enables the normally impermeant dye to bind to all DNA in the assay well. Herein, we detail our efforts to characterize and multiplex this dye when applied to suspension and adherent cell lines treated with agents which cause either primary or secondary necrosis in a 72hr time course. 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 3885. doi:1538-7445.AM2012-3885