Bioluminescent assays for high-throughput screening

In the development of high throughput screening (HTS) as a central paradigm of drug discovery, fluorescence has generally been adopted as the favored methodology. Nevertheless, luminescence has maintained a prominent position among certain assay formats, most notably genetic reporters. Recently, there has been growing partiality for luminescent assays across a wider range of applications due to their sensitivity, broad linearity, and robustness to library compounds and complex biological samples. This trend has been fostered by the development of several new assay designs for diverse targets such as kinases, cytochrome p450s, proteases, apoptosis, and cytotoxicity. This review addresses recent progress made in the use of bioluminescent assays for HTS, highlighting new detection capabilities brought about by engineering luciferase genes, enzymes, and substrates. In genetic reporter applications, modifications to the luciferase genes have improved assay sensitivity by substantially increasing expression efficiency and enhanced response dynamics by reducing expression lifetime. The performance of assays based on detection of ATP and luciferin has been enhanced by modifications to the luciferase enzyme that increase its chemical and physical stability. Detection of ATP allows rapid analysis of cell metabolism and enzymatic processes coupled to ATP metabolism. Because luciferins are not naturally associated with mammalian physiology, assays for luciferin detection utilize synthetic derivatives designed to yield luminescence only when coupled with specific target enzymes. Finally, new methods for modulating the specific activity of luciferases are leading to the development of intracellular biosensors for dynamic detection of physiological processes.     

Scintillation proximity assay in lead discovery

Background: Scintillation proximity assay (SPA) is a homogeneous scintillant bead-based platform for the measurement of biological processes and plays an important role in the identification of active chemical entities in drug discovery. Objective: The design and development of solid-phase SPA approaches are examined and compared with alternative non-radiometric fluorescence-based technologies. Methods: This review provides background on the principle of SPA and its application to biomolecular interactions from a variety of biological sources. Conclusion: The SPA approach is well suited to the demands of commercial high volume automation and assay miniaturization for target-based high-throughput screening campaigns on synthetic and natural product libraries as well as for benchtop characterization and confirmation studies. In the near future, innovations in the way SPA and fluorescence-based screening strategies are multiplexed will improve our comprehensive understanding of cellular system biology and dramatically advance the lead discovery process for the treatment of complex target-related disorders.     

Advances in functional assays for high-throughput screening of ion channels targets

Importance of the field: Ion channels are important targets for many disease areas but are challenging to screen due to lack of technologies enabling robust high-throughput assays, particularly for state-dependent interactions. Areas covered in this review: Current assay technologies used to measure ion channel function are reviewed and assessed for use in high-throughput screening (HTS). An iterative approach to screening is evaluated as an alternative to full collection screening in order to take advantage of low-throughput, high cost assays that yield high quality data. What the reader will gain: The reader will gain an understanding of the advantages and disadvantages of various assay techniques used to screen ion channels and their suitability for use in HTS. Take home message: Assays that directly measure ion channel function are prone to less artifact and higher hit confirmation in screening than those using an indirect measure but they are usually lower throughput. However, an iterative approach to screening can make the relatively lower throughput techniques amenable for use in interrogating large collections of compounds.     

Development of a complex scintillation proximity assay for highthroughput screening of PPARγmodulators

Aim: To develop a complex high-throughput screening (HTS) assay based on scintillation proximity assay (SPA) technology for identification of novel peroxisome proliferator-activated receptor gamma (PPARγ) modulators. Methods: Fulllength PPARγand retinoid X receptor alpha (RXRα), biotinylated PPAR response element (PPRE), [^3H]BRL49653 and streptavidin-coated FlashPlate or microbead were used to develop an HTS assay based on SPA technology. This ‘ABCDE‘ method was validated against conventional hydroxyapatite (HA) assay and applied to large-scale screening of 16 000 synthetic compounds and natural product extracts. Results: (1) IC50 values of positive control compounds (BRL49653 and troglitazone) obtained from the ‘ABCDE‘ method and HA assay were comparable and consistent with those reported elsewhere; (2) Approximately 178 compounds, showing more than 70% competitive inhibition on BRL49653 binding to PPARγ, were identified initially by the ‘ABCDE‘ method (microbead); (3) Secondary screening using FlashPlate and cross-reactivity studies with RARα, β,γand RXRα,β,γconfirmed that 12 compounds possessed specific PPARγbinding properties including 2 with IC50 values less than 0.5μmol/L and novel chemical structures. Conclusions: The ‘ABCDE‘ method using either FlashPlate or microbead, is a highly efficient, automatable, and robust tool to screen potential PPARγmodulators in HTS setting. Its application may be expanded to other nuclear receptors that form heterodimers upon activation.     

Scintillation proximity radioimmunoassay for the measurement of acyclovir

A homogeneous, single-tube scintillation proximity radioimmunoassay (SPRIA) to quantitate acyclovir (Zovirax^®), ACV, (9-[(2[hydroxyethoxy)]methylguanine)} in human plasma is described. The reagents for the SPRIA are an anti-ACV monoclonal antibody (WACO4 MAb), tritiated ACV, and scintillation proximity reagent (goat anti-mouse immunoglobulin G (IgG) coupled to fluoromicrospheres). The ACV standard curve range in the SPRIA is from 0.7 ng ml⁻¹ (3.0 nmol l⁻¹) to 90.0 ng ml⁻¹ (0.4 μmol l⁻¹) with a 50% inhibitory concentration of 5.0 ng ml⁻¹ (22.2 nmol l⁻¹). However, the lower limit of quantification is 7 ng ml⁻¹ at 1:10 dilution of plasma. Analytical recovery of ACV in spiked human plasma controls ranges between 90–110%. Intra- and inter-assay relative standard deviations were < 8%. This high throughput homogeneous assay is a rapid, convenient and simple alternative to the current radioimmunoassay that uses ammonium sulfate precipitation as the separation method. This technique is particularly attractive because it requires neither separation of bound from free drug nor use of scintillation fluid. The procedure was applied to quantitate ACV in samples from pre-clinical and clinical studies after the administration of valaciclovir, a prodrug of ACV (256U87, Valtrex^®, l-valyl ester of ACV). Automation of this assay will further improve efficiency in processing a larger number of samples.     

Yeast: bridging the gap between phenotypic and biochemical assays for high-throughput screening

ABSTRACT

Introduction: Both in vitro biochemical and phenotypic assay platforms have clear limitations in high throughput screening (HTS) for drug discovery. The use of genetically tractable model yeast as a vehicle for target-based HTS overcomes many of these by allowing the identification of on-target compounds that function within a eukaryotic cellular context.

Areas covered: In this special report, the use of yeast-based assays in HTS is discussed with reference to the various platforms that have been utilized over the past 20 years. The specific issues considered are the necessity to employ counter and secondary screening approaches to ensure the on-target activity of hits, and the recent developments in detection systems that have facilitated miniaturization and ultra-HTS.

Expert opinion: It is difficult at present to predict the future. That being said, the demonstrable possibilities of optimizing yeast-based HTS, coupled with the demonstration of utility in an industrial setting, shows that these platforms have the potential to bridge the gap between phenotypic and biochemical assays for HTS.     

Microbioreactors for high-throughput cytotoxicity assays

Cell culture plays a fundamental role in the biotechnology and pharmaceutical industries, impacting both drug discovery and manufacturing as well as regenerative medicine. In drug discovery, cell-based assays are increasingly being used for drug target validation and drug ADMET (absorption, distribution, metabolism, elimination and toxicity) studies because cells can provide more representative responses to drugs than simple molecular assays and are easier to use in a high-throughput format than animals. There are, however, intrinsic drawbacks associated with conventional in vitro cellular tests using two-dimensional cultures, in that they lack a three-dimensional (3D) scaffold to support cell growth and proper tissue function, and cannot mimic in vivo cellular conditions. Tailoring scaffold properties for 3D cell cultures is therefore essential in developing a representative in vitro tissue model for cytotoxicity assays. Recently, microfluidic bioreactors with miniaturized culturing vessels and high controllability for operation and on-line monitoring/sensing have gained popularity in bioprocess development and cell-based assays. The advancement in this field has been enabled by the development of novel cell lines and reporter gene techniques, as well as new microfabrication, microfluidics and optical and electrochemical sensor technologies. Non-invasive detection methods using reporter genes and label-free techniques allow for real-time dynamic monitoring of viable cell number and cellular activities. Microbioreactors with continuous perfusion allow for long-term culturing to study chronic toxicity effects. Systemic toxicity and interactions between different cell types can also be studied on a biochip. High-density microfluidic arrays provide a platform for future high-throughput and high-content screening that will contribute to drug discovery and bioprocess development.     

Advanced screening assays to rapidly identify solubility-enhancing formulations: high-throughput, miniaturization and automation

Development of solubility-enhancing formulations for poorly water-soluble compounds always poses a challenge. Conventional formulation screening assays are potentially time-consuming and labor-intensive and, moreover, require a large amount of a compound; they are not ideal when compound availability and testing time are limited. In recent years, in-vitro screening assays that are rapid, inexpensive, minimally labor-intensive, and require only small quantities of a compound have become available. These advanced assays allow high-throughput automation, miniaturization, and parallel processing, thereby enabling scientists to rapidly identify solubility-enhancing formulations with milligram or sub-milligram quantities of an active pharmaceutical ingredient (API). This article reviews these assays for rapidly screening the aqueous solubility of lead compounds and the solubility-enhancing formulations with limited quantities of API.     

Emerging trends in high-throughput screening

Novel technologies are emerging for high-throughput screening, driven by the needs and fine-tuning of established drug discovery activities, as well as by the emergence of novel target classes resulting from the deciphering of the human genome. Disciplines other than biology have now entered the screening scene, as bioinformatics, micro-technology and analytics provide powerful methodologies and applications that were not previously suitable for high-throughput screening. Many of these will move high-throughput screening from a numbers game to a content- and information-based approach to identify leads for novel disease targets.     

Novel trends in high-throughput screening

Lead discovery by high-throughput screening (HTS) has evolved into a mature scientific discipline in modern drug discovery since its beginning about 10–15 years ago. Owing to the strong efforts in automation and miniaturization, even relatively large compound collections of over one million compounds or more can be screened against a large number of biological targets in relatively short time and at relatively low cost compared to the efforts of just 5 or 10 years ago. This was only possible with the concomitant development of high-quality readout technologies for highly miniaturized screening. Whereas most of the conventional drug targets can be approached via current HTS-readout technologies, the challenge goes toward the hitherto non-tractable families of drug targets. Future trends will focus strongly toward these novel target classes such as ion channels, transporters, protein–protein interactions, among many others. It will be essential to make proper readout technologies and adequate chemical libraries available for these target classes. Chemical libraries derived from natural products, but also derived from combinatorial chemistry and automated synthesis will be a key prerequisite for success in the field, as long as enough diversity and drug-like properties are included in these chemical libraries [25]. The proper readout technologies for screening of large chemical libraries have seen strong advances in recent years [^{[2], [7] and [22••]}], nevertheless none of these technologies is void of artifacts, in particular artifacts derived from the inherent physical nature of chemical compounds in aqueous buffer [11^••]. We therefore propose that future lead discovery should pay more attention toward unambiguous identification of these compound related artifacts and toward efficient removal of these false-positive compounds from the HTS hit-lists. We strongly recommend the use of biophysical and enzymological studies in the HTS hit-list follow-up phase (‘hit validation’) in order to deliver information of the highest possible quality for subsequent hit-to-lead studies. Finally, the science and art of HTS has evolved in various phases from its beginning in the early 1990s toward today's state-of-the-art operation in lead discovery. During these 15 years, one can distinguish three phases (‘generations’) of HTS operations: during the first phase, HTS has been just the same as laboratory screening, albeit at much larger capacity; in the second phase (‘second generation HTS’), HTS has evolved toward more sophisticated assay development/adaptation, more toward dedicated tool production, but also more toward counter-screening and hit-list follow-up; in the current phase (‘third generation HTS’) we see much more flexibility with regards to the applied processes for lead discovery, a stronger focus on quality and validation of the obtained results and a better awareness for choosing a proper lead finding strategy in a target-by-target specific manner.Taken together, we can conclude that better flexibility and creativity, more quality and the use of project-related, tailor-made lead finding strategies in the discovery process will become the key drivers for the successful application of high-throughput screening in the Pharmaceutical, Biotech, and Academic drug discovery programs of the future.

References and recommended reading

Papers of particular interest, published within the period of review, have been highlighted as:

• of special interest

•• of outstanding interest

Non-stoichiometric inhibition in biochemical high-throughput screening

Introduction: Over the last 2 decades, high-throughput screening (HTS) has become one of the key strategies for the generation of new leads. Non-stoichiometric inhibition is one of the most extensively studied mechanisms responsible for the large percentage of hit compounds from biochemical screens that cannot be developed into leads. Therefore, HTS hit lists need to be sorted rapidly and efficiently into stoichiometrically binding inhibitors and compounds that affect enzyme activity non-stoichiometrically. Areas covered: This article explores the non-stoichiometric inhibition of enzymatic activity in biochemical screens, particularly by compound aggregation, and the authors explain the terminology they use to describe such compound behavior. The paper then provides a short historical overview of both academic and industrial research on compound aggregation specifically. Finally, the article discusses the implications for industrial drug discovery and the measures that can be taken to identify non-stoichiometric and aggregating inhibitors early in this process. Expert opinion: The most pragmatic approach in a lead finding campaign is to focus on the early identification of selective and stoichiometric inhibitors. The combination of multiple approaches (assessing both activity and binding) allows the enrichment of stoichiometric inhibitors at each stage of the flowchart.     

Non-stoichiometric inhibition in biochemical high-throughput screening

Introduction: Over the last 2 decades, high-throughput screening (HTS) has become one of the key strategies for the generation of new leads. Non-stoichiometric inhibition is one of the most extensively studied mechanisms responsible for the large percentage of hit compounds from biochemical screens that cannot be developed into leads. Therefore, HTS hit lists need to be sorted rapidly and efficiently into stoichiometrically binding inhibitors and compounds that affect enzyme activity non-stoichiometrically.

Areas covered: This article explores the non-stoichiometric inhibition of enzymatic activity in biochemical screens, particularly by compound aggregation, and the authors explain the terminology they use to describe such compound behavior. The paper then provides a short historical overview of both academic and industrial research on compound aggregation specifically. Finally, the article discusses the implications for industrial drug discovery and the measures that can be taken to identify non-stoichiometric and aggregating inhibitors early in this process.

Expert opinion: The most pragmatic approach in a lead finding campaign is to focus on the early identification of selective and stoichiometric inhibitors. The combination of multiple approaches (assessing both activity and binding) allows the enrichment of stoichiometric inhibitors at each stage of the flowchart.     

A fully automated [35S]GTPgammaS scintillation proximity assay for the high-throughput screening of Gi-linked G protein-coupled receptors

The diversity of physiological functions mediated by the GPCR superfamily provides a rich source of molecular targets for drug discovery programs. Consequently, a variety of assays have been designed to identify lead molecules based on ligand binding or receptor function. In one of these, the binding of [(35)S]GTPgammaS, a nonhydrolyzable analogue of GTP, to receptor-activated G-protein alpha subunits represents a unique functional assay for GPCRs and is well suited for use with automated HTS. Here we compare [(35)S]GTPgammaS scintillation proximity binding assays for two different G(i)-coupled GPCRs, and describe their implementation with automated high-throughput systems.     

Impact of high-throughput screening in biomedical research

High-throughput screening (HTS) has been postulated in several quarters to be a contributory factor to the decline in productivity in the pharmaceutical industry. Moreover, it has been blamed for stifling the creativity that drug discovery demands. In this article, we aim to dispel these myths and present the case for the use of HTS as part of a proven scientific tool kit, the wider use of which is essential for the discovery of new chemotypes.     

Sense and sensibility: the use of cell death biomarker assays in high-throughput anticancer drug screening and monitoring treatment responses

Cell-based screening allows identification of biologically active compounds, for example, potential anticancer drugs. In this review, various screening assays are discussed in terms of what they measure and how this affects interpretation and relevance. High-throughput (HT) assays of viability based on the reduction of exogenous substrates do not always reflect viability or cell number levels. Membrane integrity assays can be used for HT quantification of cell death, but are non-specific as to the death mode. Several HT assays monitor end point apoptosis. Screening libraries at a single concentration (micromolar) can prevent detection of potent apoptosis inducers, as high concentrations may induce mainly necrosis. Using monolayer cultures limits the significance of cell-based screening as the properties of monolayer cells differ from tumours in vivo. Spheroid cultures are more physiological, but are impractical for screening by conventional methods. The authors have developed an assay quantifying accumulation of a caspase-cleaved protein specific for epithelial cells. It provides an integrated measure of apoptosis in two- and three-dimensional cultures and can be used as a blood biomarker assay for tumour apoptosis in vivo.     

Integration of virtual and high-throughput screening

High-throughput and virtual screening are important components of modern drug discovery research. Typically, these screening technologies are considered distinct approaches, as one is experimental and the other is theoretical in nature. However, given their similar tasks and goals, these approaches are much more complementary to each other than often thought. Various statistical, informatics and filtering methods have recently been introduced to foster the integration of experimental and in silico screening and maximize their output in drug discovery. Although many of these ideas and efforts have not yet proceeded much beyond the conceptual level, there are several success stories and good indications that early-stage drug discovery will benefit greatly from a more unified and knowledge-based approach to biological screening, despite the many technical advances towards even higher throughput that are made in the screening arena.