A dual-readout F2 assay that combines fluorescence resonance energy transfer and fluorescence polarization for monitoring bimolecular interactions

F?rster (fluorescence) resonance energy transfer (FRET) and fluorescence polarization (FP) are widely used technologies for monitoring bimolecular interactions and have been extensively used in high-throughput screening (HTS) for probe and drug discovery. Despite their popularity in HTS, it has been recognized that different assay technologies may generate different hit lists for the same biochemical interaction. Due to the high cost of large-scale HTS campaigns, one has to make a critical choice to employee one assay platform for a particular HTS. Here we report the design and development of a dual-readout HTS assay that combines two assay technologies into one system using the Mcl-1 and Noxa BH3 peptide interaction as a model system. In this system, both FP and FRET signals were simultaneously monitored from one reaction, which is termed "Dual-Readout F(2) assay" with F(2) for FP and FRET. This dual-readout technology has been optimized in a 1,536-well ultra-HTS format for the discovery of Mcl-1 protein inhibitors and achieved a robust performance. This F(2) assay was further validated by screening a library of 102,255 compounds. As two assay platforms are utilized for the same target simultaneously, hit information is enriched without increasing the screening cost. This strategy can be generally extended to other FP-based assays and is expected to enrich primary HTS information and enhance the hit quality of HTS campaigns.     

Tools for GPCR drug discovery

G-protein-coupled receptors (GPCRs) mediate many important physiological functions and are considered as one of the most successful therapeutic targets for a broad spectrum of diseases. The design and implementation of high-throughput GPCR assays that allow the cost-effective screening of large compound libraries to identify novel drug candidates are critical in early drug discovery. Early functional GPCR assays depend primarily on the measurement of G-protein-mediated 2nd messenger generation. Taking advantage of the continuously deepening understanding of GPCR signal transduction, many G-protein-independent pathways are utilized to detect the activity of GPCRs, and may provide additional information on functional selectivity of candidate compounds. With the combination of automated imaging systems and label-free detection systems, such assays are now suitable for high-throughput screening (HTS). In this review, we summarize the most widely used GPCR assays and recent advances in HTS technologies for GPCR drug discovery.     

Optimizing the hit-to-lead process using SPR analysis

Secondary screening and lead optimization, where a large number of "hit" compounds are refined to a viable set of "lead" drug candidates, are considered to be bottlenecks to the drug discovery process and are targets for streamlining. Surface plasmon resonance (SPR) is a nonlabel technology that can generate kinetic data on biomolecular interactions. This allows researchers to quantitate the binding characteristics of lead compounds with their targets in terms of affinity, specificity, and association/dissociation rates in parallel. The latest generation of SPR biosensors integrate the hit-to-lead process and generate a greater depth of information, providing answers that cannot be addressed by traditional end-point assays. This allows users to make more informed choices on the selection of candidate molecules prior to preclinical development. A number of studies have used SPR biosensors in secondary screening, lead optimization, quantitative structure-activity relationship analysis, and predictive adsorption, distribution, metabolism, excretion, and/or toxicity evaluations.     

Application of NMR screening in drug discovery

The application of NMR screening in drug discovery has recently attained heightened importance throughout the pharmaceutical industry. NMR screening can be applied at various points in a drug discovery program, ranging from very early in the program, when new targets can be screened long before an HTS enzymatic assay is developed, to later in the program, as in the case where no useful hits have been detected by HTS using biological assays. The binders determined in primary NMR screens are used to guide secondary screens, which can be either completely NMR driven or use NMR in combination with other biophysical techniques. In this review we briefly discuss the methods and techniques used in NMR screening. Then, we describe in detail the NMR screening strategies and their applications to specific targets, including successful examples from actual drug design programs at our own and other pharmaceutical companies.     

SPR-based fragment screening: advantages and applications

Fragment-based screening has recently evolved into a promising strategy in drug discovery, and a range of biophysical methods can be employed for fragment library screening. Relevant approaches, such as X-ray, NMR and tethering are briefly introduced focussing on their suitability for fragment-based drug discovery. In particular the application of surface plasmon resonance (SPR) techniques to the primary screening of large libraries comprising small molecules is discussed in detail. SPR is known to be a powerful tool for studying biomolecular interactions in a sensitive and label-free detection format. Advantages of SPR methods over more traditional assay formats are discussed and the application of available channel and array based SPR systems to biosensing are reviewed. Today, SPR protocols have been applied to secondary screening of compound libraries and hit conformation, but primary screening of large fragment libraries for drug discovery is often hampered by the throughput of available systems. Chemical microarrays, in combination with SPR imaging, can simultaneously generate affinity data for protein targets with up to 9,216 immobilized fragments per array. This approach has proven to be suitable for screening fragment libraries of up to 110,000 compounds in a high throughput fashion. The design of fragment libraries and appropriate immobilization chemistries are discussed, as well as suitable follow-up strategies for fragment hit optimization. Finally, described case studies demonstrate the successful identification of selective low molecular weight inhibitors for pharmacologically relevant drug targets through the SPR screening of fragment libraries.     

Phage display for target-based antibacterial drug discovery

Increasing bacterial drug resistance and hard-to-eradicate opportunistic infections have created a need for new antibiotics. Sequencing of microbial genomes has yielded many new potential targets for antibacterial drug discovery. However, little is known about the biochemical activities of many of these targets, making it difficult to develop HTS assays for them. Peptides isolated by phage display can be used as ‘surrogate ligands’ in competition assays for screening of targets of unknown function with small-molecule libraries. These screening assays can be adapted into a variety of high-throughput formats, including those based on radioactive, luminescence or fluorescence detection.     

High throughput screening assay for UDP-glucuronosyltransferase 1A1 glucuronidation profiling

Development of high throughput screening (HTS) assays for evaluation of a compound's toxicity and potential for drug-drug interactions is a critical step towards production of better drug candidates and cost reduction in the drug development process. HTS assays for drug metabolism mediated by cytochrome P450s are now routinely used in compound library characterization and for computer modeling studies. However, development and application of HTS assays involving UDP-glucuronosyltransferases (UGTs) are lagging behind. Here we describe the development of a fluorescence-based HTS assay for UGT1A1 using recombinant enzyme and fluorescent substrate in the presence of an aqueous solution of PreserveX-QML (QBI Life Sciences, Madison, WI) polymeric micelles, acting as a stabilizer and a blocker of nonspecific interactions. The data include assay characteristics in 384-well plate format obtained with robotic liquid handling equipment and structures of hits (assay modifiers) obtained from the screening of a small molecule library at the University of Wisconsin HTS screening facility. The application of the assay for predicting UGT-related drug-drug interactions and building pharmacophore models, as well as the effects of polymeric micelles on the assay performance and compound promiscuity, is discussed.     

High-throughput screening technologies for drug glucuronidation profiling

A significant number of endogenous and exogenous compounds, including many therapeutic agents, are metabolized in humans via glucuronidation, catalysed by uridine diphosphoglucuronosyltransferases (UGTs). The study of the UGTs is a growing field of research, with constantly accumulated and updated information regarding UGT structure, purification, substrate specificity and inhibition, including clinically relevant drug interactions. Development of reliable UGT assays for the assessment of individual isoform substrate specificity and for the discovery of novel isoform-specific substrates and inhibitors is crucial for understanding the function and regulation of the UGT enzyme family and its clinical and pharmacological relevance. High-throughput screening (HTS) is a powerful technology used to search for novel substrates and inhibitors for a wide variety of targets. However, application of HTS in the context of UGTs is complicated because of the poor stability, low levels of expression, low affinity and broad substrate specificity of the enzymes, combined with difficulties in obtaining individual UGT isoforms in purified format, and insufficient information regarding isoform-specific substrates and inhibitors. This review examines the current status of HTS assays used in the search for novel UGT substrates and inhibitors, emphasizing advancements and challenges in HTS technologies for drug glucuronidation profiling, and discusses possible avenues for future advancement of the field.     

Design of small molecule libraries for NMR screening and other applications in drug discovery

There are conceptual differences between high-throughput screening (HTS) and fragment-based screening by NMR. The number of compounds in libraries for NMR screening may be significantly smaller than those used for HTS. Because one relies on a small library its design is significantly important and is the object of this article. A short introduction on fragment-based NMR screening approaches will be provided. Although there are currently very few reports describing the design of libraries of small molecules for NMR screening, aspects of the question of how to compile diverse collections of small molecular fragments useful for drug design were previously addressed for the purposes of combinatorial library design and de novo drug design. As these disciplines are highly interrelated and are applied in an interconnected manner with NMR screening within the drug discovery process, a review of combinatorial library design and especially the building block or fragment selection strategies applied for combinatorial library design and de novo design is well suited to reveal fundamental strategies and potential techniques for the design of NMR screening libraries. This section will be rounded off by a report on hands-on-experience with the design of the Novartis second-site NMR screening library and practical considerations for the design of compound mixtures. Rather than providing an exact protocol general guidelines will be indicated.     

Scintillation proximity assays in high-throughput screening

Scintillation proximity assays (SPAs) have become a powerful tool for high-throughput screening (HTS) because they can measure the activity and binding of very diverse classes of drug targets. By applying the basic principles of ligand-receptor binding and enzyme kinetics, it is possible to build a large variety of miniaturized, high-throughput assays and screen millions of compounds. SPAs are enabled by the diversity of radiolabeled molecules and affinity tags that are commercially available. These synthetic radiotracers allow for minimal disturbance of the natural binding interactions. This article will present a comprehensive review of the technique and provide detailed information on its applications related to HTS, highlighting the major uses and giving some suggestions for future research.     

Current in vitro kinase assay technologies: the quest for a universal format

The rapidly growing interest in kinases as drug targets has prompted the development of many kinase assay technologies. These technologies can be grouped into three categories: radiometric assays, phospho-antibody-dependent fluorescence/luminescence assays, and phospho-antibody-independent fluorescence/luminescence assays. This article will review some of the major kinase assay technologies on the market, with particular emphasis on the newest systems. We will describe the physical principles, the practical advantages and drawbacks, and the potential applications of these technologies in kinase drug discovery. Most of these technologies are suitable for HTS, but only a few can be utilized for kinetic and mechanistic studies. Significant progress towards development of generic assays, free of radioisotopes and custom reagents such as phospho-specific antibodies, has been made in recent years. However, due to various limitations of each format, none of these generic assay technologies can yet claim to be truly universal. Several factors, including the intended applications, cost, timeline, expertise, familiarity, and comfort level, should be considered prior to pursuing a particular kinase assay technology.     

Cellular HTS assays for pharmacological characterization of Na(V)1.7 modulators

Ion channels are challenging targets in the early phases of the drug discovery process, especially because of the lack of technologies available to screen large numbers of compounds in functionally relevant assays. The electrophysiological patch-clamp technique, which is the gold standard for studying ion channels, has low throughput and is not amenable to screening large numbers of compounds. However, for random high-throughput screening (HTS) of compounds against ion channel targets, a number of functional cellular assays have become available during the last few years. Here we use the sodium channel NaV1.7 stably expressed in human embryonic kidney 293 cells and compare three HTS assays-a Li flux atomic absorption spectroscopy (AAS) assay, a fluorescent imaging plate reader (FLIP, Molecular Devices, Sunnyvale, CA) membrane potential assay, and a fluorescence resonance energy transfer (FRET)-based membrane potential assay-to an automated electrophysiological assay (the Ionworks HT [Molecular Devices] platform) and characterize 11 known NaV inhibitors. Our results show that all three HTS assays are suitable for identification of NaV1.7 inhibitors, but as an HTS assay the Li-AAS assay is more robust with higher Z' values than the FLIPR and FRET-based membrane potential assays. Furthermore, there was a better correlation between the Ionworks assay and the Li-AAS assay regarding the potency of the NaV inhibitors investigated. This paper describes the first comparison between all the HTS assays available today to study voltage-gated NaVs, and the results suggest that the Li-AAS assay is more suited as a first HTS assay when starting an NaV drug discovery campaign.     

G-蛋白偶联受体的功能测定和高通量药物筛选

G 蛋白偶联受体家族是药物开发中最大的一类药物靶点 ,高通量药物筛选是开发药物早期阶段的最重要工具之一。根据G 蛋白偶联受体与配体结合及激发的信号通路 ,人们设计了各种可行的功能测试方法 ,用于G 蛋白偶联受体为药靶的高通量药物筛选 ,如 :微体积荧光数字图像测定技术 (Fluorometicmicrovolumeassaytechnology ,FMAT)、荧光偏振 (Fluoresencepolarization ,FP)、竞争性ELISA (Com petitiveenzyme linkedimmunosorbent)、闪烁邻近测定法(Scintillation proximityassay ,SPA)、载黑色素细胞测定法(Melanophoreassay)、报告基因测定法 (Reportergeneassay)和钙离子测定法等测定方法。在这些方法中 ,报告基因测定法和钙离子测定法占了主导地位。非放射性、无需底物和辅助剂的报告基因测定方法和荧光钙离子指示剂的钙离子测定方法可能是将来G 蛋白偶联受体的功能分析和高通量药物筛选的发展方向     

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.     

How can high-throughput screening deliver drugs to treat atherosclerosis?

Importance of the field: Atherosclerosis is a progressive disease that is characterized by the accumulation of lipid-rich plaques within the artery walls. Despite the past 3 decades witnessing the most significant advances in the pharmacotherapy of atherosclerosis with statins, atherosclerosis is still one of the leading causes of mortality in industrialized and developing nations. The applications of high-throughput screening (HTS) have retrieved hits and lead compounds which may be further developed to new promising therapeutics to achieve more effective reductions in the risk of cardiovascular morbidity and mortality. Areas covered in this review: The review provides a summary of potential drug targets other than HMG-CoA reductase (primary target of statins) and their application in biochemical or cell-based HTS assays used by pharmaceutical companies and academic laboratories for anti-atherosclerotic drug discovery. What the reader will gain: The reader will gain an overview of the HTS strategies currently used in the development of anti-atherosclerotic agents. The reader is also provided with some abortive examples in anti-atherosclerotic drug discovery as well as the associated limitations and challenges of the process that HTS delivers new drugs to treat atherosclerosis. Take home message: HTS can assist in the efficient discovery of new drugs towards the potential targets involved in the progress of atherosclerosis.     

Virtual screening and its integration with modern drug design technologies

Drug discovery is a highly complex and costly process, which demands integrated efforts in several relevant aspects involving innovation, knowledge, information, technologies, expertise, R&D investments and management skills. The shift from traditional to genomics- and proteomics-based drug research has fundamentally transformed key R&D strategies in the pharmaceutical industry addressed to the design of new chemical entities as drug candidates against a variety of biological targets. Therefore, drug discovery has moved toward more rational strategies based on our increasing understanding of the fundamental principles of protein-ligand interactions. The combination of available knowledge of several 3D protein structures with hundreds of thousands of small-molecules have attracted the attention of scientists from all over the world for the application of structure- and ligand-based drug design approaches. In this context, virtual screening technologies have largely enhanced the impact of computational methods applied to chemistry and biology and the goal of applying such methods is to reduce large compound databases and to select a limited number of promising candidates for drug design. This review provides a perspective of the utility of virtual screening in drug design and its integration with other important drug discovery technologies such as high-throughput screening (HTS) and QSAR, highlighting the present challenges, limitations, and future perspectives in medicinal chemistry.     

Strategies and techniques for identification of novel bioactive compounds--CHI's second annual conference. 7-9 October 1998, Zurich, Switzerland

This conference on recent developments in the discovery of novel therapeutic candidates was organized by Amy Dasch (Cambridge Healthtech Institute, Newton Upper Falls, MA, USA; http://www.xensei.com/conferences). The conference provided an overview of all relevant aspects of the rapidly changing paradigms in drug research. Gene technology creates a vast number of new biological targets. The progress in combinatorial chemistry and high-throughput screening (HTS) is accompanied by the development of virtual libraries, large screening programs, and the generation of enormous sets of data. Correspondingly, the lectures covered such different topics as target identification and assay development, HTS technology, combinatorial library design and synthesis, chemoinformatics, and the integration of these components into the discovery of novel pharmaceutical compounds, the development of agricultural chemicals, and other applications. A most valuable addition was reports on case histories in drug development from pharmaceutical companies utilizing these technologies. About 100 scientists, many of them from European countries, attended the meeting. In total, 25 lectures were presented in four sessions: molecular diversity and library design; combinatorial synthesis; HTS; computational methodologies and chemoinformatics. Like other commercially organized conferences, this meeting was well-planned. The balance of speakers from small venture capital companies, large pharmaceutical and agricultural firms gave a broad overview of recent progress in the rational design, combinatorial synthesis, and HTS of new bioactive compounds, as well as on different approaches to handling large data sets and deriving structure-activity relationships from such data.     

Design of chemical libraries for screening

Importance of the field: The ultimate goal of discovery screening is to have a fast and cost-effective strategy to meet the demands of producing high-content lead series with improved prospects for clinical success. While high-throughput screening (HTS) dominates the drug discovery landscape, other processes and technologies have emerged, including high-content screening and fragment-based design to provide alternatives that may be more suitable for certain targets. There has been a growing interest in reducing the number of compounds to be screened to prevent the escalation in the costs, time and resources associated with HTS campaigns. Library design plays a central role in these efforts.

Areas covered in this review: This opinion provides a survey of some recent developments in the diversity based library design process, but within a historical context. In particular, the importance of chemotyping and substructure analysis and the challenges presented by novel lead discovery technologies that require the design of libraries for screening are discussed.

What the reader will gain: Readers will gain an appreciation of some developments in the field of library design and the factors that are driving the development of new library design technologies; specifically, challenges presented for chemoinformatics with the novel screening technologies in diversity based screening and compound filtering.

Take home message: Chemotyping and substrutural analysis are techniques that have been underutilized in the process of library design. However, they offer a direct way to evaluate libraries and have been successfully used to develop predictive methodologies. Tools are available to this end, but the full power of the approach has not been realized yet.     

Aptamers as tools for target prioritization and lead identification

The increasing number of potential drug target candidates has driven the development of novel technologies designed to identify functionally important targets and enhance the subsequent lead discovery process. Highly specific synthetic nucleic acid ligands – also known as aptamers – offer a new exciting route in the drug discovery process by linking target validation directly with HTS. Recently, aptamers have proven to be valuable tools for modulating the function of endogenous cellular proteins in their natural environment. A set of technologies has been developed to use these sophisticated ligands for the validation of potential drug targets in disease models. Moreover, aptamers that are specific antagonists of protein function can act as substitute interaction partners in HTS assays to facilitate the identification of small-molecule lead compounds.