Andrew L. Niles

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.

Target engagement and drug residence time can be observed in living cells with BRET

The therapeutic action of drugs is predicated on their physical engagement with cellular targets. Here we describe a broadly applicable method using bioluminescence resonance energy transfer (BRET) to reveal the binding characteristics of a drug with selected targets within intact cells. Cell-permeable fluorescent tracers are used in a competitive binding format to quantify drug engagement with the target proteins fused to Nanoluc luciferase. The approach enabled us to profile isozyme-specific engagement and binding kinetics for a panel of histone deacetylase (HDAC) inhibitors. Our analysis was directed particularly to the clinically approved prodrug FK228 (Istodax/Romidepsin) because of its unique and largely unexplained mechanism of sustained intracellular action. Analysis of the binding kinetics by BRET revealed remarkably long intracellular residence times for FK228 at HDAC1, explaining the protracted intracellular behaviour of this prodrug. Our results demonstrate a novel application of BRET for assessing target engagement within the complex milieu of the intracellular environment.

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.

Recombinant human mast cell tryptase beta: stable expression in Pichia pastoris and purification of fully active enzyme

Human mast cell tryptase beta (EC 3.4.21.59) is a trypsin‐like serine protease that is stored in and released from mast cell granules. This enzyme has been expressed in Pichia pastoris via homologous recombination of the cDNA coding for the mature active tryptase with the addition of a KEX 2 processing site into the Pichia genome. Cells producing recombinant human tryptase (rHT) were selected by screening with antibodies. Induction with methanol resulted in the secretion of rHT into the Pichia growth medium; tryptase activity was stabilized by the addition of heparin to the culture medium. Increasing levels of enzyme were detected in the medium for up to 3 days. Fully active enzyme was purified from the culture medium with a 100% yield of activity via a simple two‐step procedure, with hydrophobic interaction chromatography followed by affinity chromatography on immobilized heparin. Bands of 33 (faint), 34.2, 35.9 and 50 kDa (diffuse) were observed on SDS/PAGE. These multiple forms were due to differences in post‐translational glycosylation of asparagine residues, because enzymic deglycosylation resulted in only one band at 33 kDa. A single symmetrical peak with an estimated size of 197 kDa was obtained on gel filtration. Kinetic analyses in comparison with native human lung mast cell tryptase (HLT) yielded similar Km values, but the kcat of rHT was more than twice that of HLT.

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.

Validation of a HTS-amenable assay to detect drug-induced mitochondrial toxicity in the absence and presence of cell death.

Drug-induced mitochondrial dysfunction is known to contribute to late stage compound attrition. Recently, assays that identify mitochondrial dysfunction have been developed but many require expensive reagents, specialized equipment, or specialized expertise such as isolation of mitochondria. Here, we validate a new 384-well format cell-based dual parameter assay that uses commonly available detection methods to measure both mitochondrial toxicity and cytotoxicity. In our initial evaluation, antimycin A, CCCP, nefazodone, flutamide, and digitonin were tested in K562 cells in both glucose- and galactose-supplemented media with a 2h incubation. The assay was able to correctly differentiate these compounds into mitochondrial toxicants and non-mitochondrial toxicants, and had excellent reproducibility. We next tested 74 compounds in K562 cells in both types of media and show that the assay was able to correctly identify some of the compounds as mitochondrial toxicants. Moreover, the assay could be simplified, without loss of information, by using K562 cells in galactose-containing medium alone. This simple, robust assay can be positioned as a rapid, early readout of mitochondrial and cellular toxicity. However, since the assay fails to identify some mitochondrial toxicants, further assays may be required to detect mitochondrial toxicity once lead compounds have been selected.

A Bioluminogenic HDAC Activity Assay: Validation and Screening

Histone deacetylase (HDAC) enzymes modify the acetylation state of histones and other important proteins. Aberrant HDAC enzyme function has been implicated in many diseases, and the discovery and development of drugs targeting these enzymes is becoming increasingly important. In this article, the authors report the evaluation of homogeneous, single-addition, bioluminogenic HDAC enzyme activity assays that offer less assay interference by compounds in comparison to fluorescence-based formats. The authors assessed the key operational assay properties including sensitivity, scalability, reproducibility, signal stability, robustness (Z′), DMSO tolerance, and pharmacological response to standard inhibitors against HDAC-1, HDAC-3/NcoR2, HDAC-6, and SIRT-1 enzymes. These assays were successfully miniaturized to a 10 µL assay volume, and their suitability for high-throughput screening was tested in validation experiments using 640 drugs approved by the Food and Drug Administration and the Hypha Discovery MycoDiverse natural products library, which is a collection of 10 049 extracts and fractions from fermentations of higher fungi and contains compounds that are of low molecular weight and wide chemical diversity. Both of these screening campaigns confirmed that the bioluminogenic assay was high-throughput screening compatible and yielded acceptable performance in confirmation, counter, and compound/extract and fraction concentration-response assays.

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.