Terry Riss

In Vitro Viability and Cytotoxicity Testing and Same-Well Multi-Parametric Combinations for High Throughput Screening

In vitro cytotoxicity testing has become an integral aspect of drug discovery because it is a convenient, costeffective, and predictive means of characterizing the toxic potential of new chemical entities. The early and routine implementation of this testing is testament to its prognostic importance for humans. Although a plethora of assay chemistries and methods exist for 96-well formats, few are practical and sufficiently sensitive enough for application in high throughput screening (HTS). Here we briefly describe a handful of the currently most robust and validated HTS assays for accurate and efficient assessment of cytotoxic risk. We also provide guidance for successful HTS implementation and discuss unique merits and detractions inherent in each method. Lastly, we discuss the advantages of combining specific HTS compatible assays into multi-parametric, same-well formats.

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.

Update on in vitro cytotoxicity assays for drug development.

Background: in vitro cytotoxicity testing provides a crucial means of ranking compounds for consideration in drug discovery. The choice of using a particular viability or cytotoxicity assay technology may be influenced by specific research goals. Objective: Although the high-throughput screening (HTS) utility is typically dependent upon sensitivity and scalability, it is also impacted by signal robustness and resiliency to assay interferences. Further consideration should be given to data quality, ease-of-use, reagent stability, and matters of cost-effectiveness. Methods: Here we focus on three main classes of assays that are at present the most popular, useful, and practical for HTS drug discovery efforts. These methods measure: i) viability by metabolism reductase activities; ii) viability by bioluminescent ATP assays; or iii) cytotoxicity by enzymes ‘released’ into culture medium. Multi-parametric technologies are also briefly discussed. Results/conclusion: Each of these methods has its relative merits and detractions; however multi-parametric methods using both viability and cytotoxicity markers may mitigate the inherent shortcomings of single parameter measures.

Bioluminescent assays for ADMET.

Bioluminescent assays couple a limiting component of a luciferase-catalyzed photon-emitting reaction to a variable parameter of interest, while holding the other components constant or non-limiting. In this way light output varies with the parameter of interest. This review describes three bioluminescent assay types that use firefly luciferase to measure properties of drugs and other xenobiotics which affect their absorption, distribution, metabolism, elimination and toxicity. First, levels of the luciferase enzyme itself are measured in gene reporter assays that place a luciferase cDNA under the control of regulatory sequences from ADMET-related genes. This approach identifies activators of nuclear receptors that regulate expression of genes encoding drug-metabolizing enzymes and drug transporters. Second, drug effects on enzyme activities are monitored with luminogenic probe substrates that are inactive derivatives of the luciferase substrate luciferin. The enzymes of interest convert the substrates to free luciferin, which is detected in a second reaction with luciferase. This approach is used with the drug-metabolizing CYP and monoamine oxidase enzymes, apoptosis-associated caspase proteases, a marker protease for non-viable cells and with glutathione-S-transferase to measure glutathione levels in cell lysates. Third, ATP concentration is monitored as a marker of cell viability or cell death and as a way of identifying substrates for the ATP-dependent drug transporter, P-glycoprotein. Luciferase activity is measured in the presence of a sample that supplies the requisite luciferase substrate, ATP, so that light output varies with ATP concentration. The bioluminescent ADMET assays are rapid and sensitive, amenable to automated high-throughput applications and offer significant advantages over alternative methods.

Cytotoxicity Testing: Measuring Viable Cells, Dead Cells, and Detecting Mechanism of Cell Death

Testing the effects of compounds on the viability of cells grown in culture is widely used as a predictor of potential toxic effects in whole animals. Among the several alternative assays available, measuring the levels of ATP is the most sensitive, reliable, and convenient method for monitoring active cell metabolism. However, recently developed combinations of methods have made it possible to collect more information from in vitro cytotoxicity assays using standard fluorescence and luminescence plate readers. This chapter describes two assay methods. The first utilizes beetle luciferase for measuring the levels of ATP as a marker of viable cells. The second more recently developed multiplex method relies on selective measurement of three different protease activities as markers for viable, necrotic, and apoptotic cells. Data analysis from the measurement of three marker protease activities from the same sample provides a useful tool to help uncover the mechanism of cell death and can serve as an internal control to help identify assay artifacts.

APOPTOSIS AS A BIOMARKER IN CHEMOPREVENTION TRIALS

Monitoring apoptosis in histologic samples is becoming increasingly important as more effort is applied to the study of therapeutic modulators of cell death in tumors. The common methods used to monitor DNA fragmentation and cell morphology as markers of apoptosis have their own set of advantages and disadvantages. Growing knowledge of the signaling events that occur during cell death has established the caspase enzymes as the central executioner proteases of apoptosis. The results of caspase activity generate neo-epitopes that occur only during apoptosis. Directly monitoring caspase-mediated events as markers of apoptosis offers advantages over existing assay methods. Recently, several new marker antibodies have been developed that detect active caspase enzymes or the products they produce. The possibility of using these new marker antibodies for monitoring prostate chemoprevention trials is presented in this review.

Multiplex Caspase Activity and Cytotoxicity Assays

Multiplexed assay chemistries provide for multiple measurements of cellular parameters within a single assay well. This experimental practice not only is more cost efficient but provides more informational content about a compound or treatment. For instance, multiplexed caspase activity assays can help establish the kinetics and magnitude of initiator and effector caspase induction by candidate compounds or treatments. The ability to combine the activity profiles within the same sample provides a level of normalization not possible with parallel assays. Furthermore, multiplexing caspase activity assays with viability and/or cytotoxicity assays can support conclusions regarding cytotoxic mechanism and provide normalization that may help correct for differences in cell number.

“Multiplexed Viability, Cytotoxicity, and Caspase Activity Assays”

Multiplexed assay chemistries provide for multiple measurements of cellular parameters within a single assay well. This experimental practice is not only more cost efficient, but also provides more information about a compound or treatment. The ability to combine the activity profiles within the same sample provides a level of normalization not possible with parallel assays. Furthermore, multiplexing caspase activity assays with viability and/or cytotoxicity assays can support conclusions regarding cytotoxic mechanism and provide normalization, which may help correct for differences in cell number.

Assay Guidance Manual: Quantitative Biology and Pharmacology in Preclinical Drug Discovery

The Assay Guidance Manual (AGM) is an eBook of best practices for the design, development, and implementation of robust assays for early drug discovery. Initiated by pharmaceutical company scientists, the manual provides guidance for designing a “testing funnel” of assays to identify genuine hits using high‐throughput screening (HTS) and advancing them through preclinical development. Combined with a workshop/tutorial component, the overall goal of the AGM is to provide a valuable resource for training translational scientists.

Abstract 3505: A bioluminescent, homogeneous annexin V microplate-based method for assessment of apoptosis

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.