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.

High-content micronucleus assay in genotoxicity profiling: initial-stage development and some applications in the investigative/lead-finding studies in drug discovery.

This article describes the first step toward full (that includes conditions for both absence and presence of metabolic activation) validation and drug discovery application of a 96-well, automated, high-content micronucleus (HCMN) assay. The current validation tests were performed using Chinese hamster ovary cells, in the absence of metabolic activation, against three distinct sets of drug-like compounds that represent all stages of a drug discovery pipeline. A compound categorization scheme was created based on quantitative relationships between micronucleus (MN) signals, cytotoxicity, and compound solubility. Results from initial validation compounds (n = 38) set the stage for differentiating overall positive and negative MN inducers. To delve deeper into the compound categorization process, a more extensive validation set, consisting of a larger set (n = 370) of "drug-like but less optimized" early-stage compounds, was used for further refinement of positive and negative compound categories. The predictivity and applicability of the assay for clinical stage compounds was ascertained using (n = 168) clinically developed marketed drugs or well-studied compounds. Upon full validation, a detailed analysis of results established five compound categories--NEG (negative), NEG/xx µM (negative up to the solubility limit of xx µM), WPOS (weak positive), POS (positive), and INCON (inconclusive). Furthermore, examples of lead-finding applications and ongoing investigative HCMN activities are described. A proposal is offered on how the HCMN assay can be positioned in parallel to the overall stage gates (e.g., scaffold selection, lead optimization, late-stage preclinical development) of drug discovery programs. Because of its greater throughput, 1-week turnaround time, and a substantially reduced (1-2 mg) requirement for compound consumption, the HCMN assay is appropriate for developing structure-genotoxicity relationships and for mechanistic genotoxicity studies. The assay does not replace the Organization for Economic Cooperation and Development-compliant, non-good laboratory practice in vitro MN test (e.g., slide-based MN test in TK6 lymphoblastoid cells) that is used for full characterization of lead candidates.

Assay development and screening of human DGAT1 inhibitors with an LC/MS-based assay: application of mass spectrometry for large-scale primary screening.

Many attractive targets for therapeutic intervention are enzymes that catalyze biological reactions involving small molecules such as lipids, fatty acids, amino acid derivatives, nucleic acid derivatives, and cofactors. Some of the reactions are difficult to detect by methods commonly used in high-throughput screening (HTS) without specific radioactive or fluorescent labeling of substrates. In addition, there are instances when labeling has a detrimental effect on the biological response. Generally, applicable assay methodologies for detection of such reactions are thus required. Mass spectrometry (MS), being a label-free detection tool, has been actively pursued for assay detection in HTS in the past several years. The authors have explored the use of multiparallel liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) for high-throughput detection of biochemical reactions. In this report, we describe in detail the assay development and screening with a LC/MS-based system for inhibitors of human diacylglycerol acyltransferase (DGAT1) with a chemical library of approximately 800,000 compounds. Several strategies and process improvements have been investigated to overcome technical challenges such as data variation and throughput. Results indicated that, through these innovative approaches, the LC/MS-based screening method is both feasible and suitable for high-throughput primary screening.

Novel trends in high-throughput screening.

High-throughput screening (HTS) is a well-established process for lead discovery in Pharma and Biotech companies and is now also being used for basic and applied research in academia. It comprises the screening of large chemical libraries for activity against biological targets via the use of automation, miniaturized assays and large-scale data analysis. Since its first advent in the early to mid 1990s, the field of HTS has seen not only a continuous change in technology and processes, but also an adaptation to various needs in lead discovery. HTS has now evolved into a mature discipline that is a crucial source of chemical starting points for drug discovery. Whereas in previous years much emphasis has been put on a steady increase in screening capacity ('quantitative increase') via automation and miniaturization, the past years have seen a much greater emphasis on content and quality ('qualitative increase'). Today, many experts in the field see HTS at a crossroad with the need to decide on either higher throughput/more experimentation or a greater focus on assays of greater physiological relevance, both of which may lead to higher productivity in pharmaceutical R&D. In this paper, we describe the development of HTS over the past decade and point out our own ideas for future directions of HTS in biomedical research. We predict that the trend toward further miniaturization will slow down with the balanced implementation of 384 well, 1536 well, and 384 low volume well plates. Furthermore, we envisage that there will be much more emphasis on rigorous assay and chemical characterization, particularly considering that novel and more difficult target classes will be pursued. In recent years we have witnessed a clear trend in the drug discovery community toward rigorous hit validation by the use of orthogonal readout technologies, label free and biophysical methodologies. We also see a trend toward a more flexible use of the various screening approaches in lead discovery, that is, the use of both full deck compound screening as well as the use of focused screening and iterative screening approaches. Moreover, we expect greater usage of target identification strategies downstream of phenotypic screening and the more effective implementation of affinity selection technologies as a result of advances in chemical diversity methodologies. We predict that, ultimately, each hit finding strategy will be much more project-related, tailor-made, and better integrated into the broader drug discovery efforts.

Evaluation of fluorescence- and mass spectrometry-based CYP inhibition assays for use in drug discovery.

The potential for metabolism-related drug-drug interactions by new chemical entities is assessed by monitoring the impact of these compounds on cytochrome P450 (CYP) activity using well-characterized CYP substrates. The conventional gold standard approach for in vitro evaluation of CYP inhibitory potential uses pooled human liver microsomes (HLM) in conjunction with prototypical drug substrates, often quantified by LC-MS/MS. However, fluorescent CYP inhibition assays, which use recombinantly expressed CYPs and fluorogenic probe substrates, have been employed in early drug discovery to provide low-cost, high-throughput assessment of new chemical entities. Despite its greatly enhanced throughput, this approach has been met with mixed success in predicting the data obtained with the conventional gold standard approach (HLM+LC-MS). The authors find that the predictivity of fluorogenic assays for the major CYP isoforms 3A4 and 2D6 may depend on the quality of the test compounds. Although the structurally more optimized marketed drugs yielded acceptable correlations between the fluorogenic and HLM+LC-MS/MS assays for CYPs 3A4, 2D6, and 2C9 (r2 = 0.5-0.7; p < 0.005), preoptimization, early discovery compounds yielded poorer correlations (r2 < or = 0.2) for 2 of these major isoforms, CYPs 3A4 and 2D6. Potential reasons for the observed differences are discussed.

Which aspects of HTS are empirically correlated with downstream success?

High-throughput screening (HTS) is a well-established hit-finding approach used in the pharmaceutical industry. In this article, recent experience at Novartis with respect to factors influencing the success of HTS campaigns is discussed. An inherent measure of HTS quality could be defined by the assay Z and Z' factors, the number of hits and their biological potencies; however, such measures of quality do not always correlate with the advancement of hits to the later stages of drug discovery. Also, for many target classes, such as kinases, it is easy to identify hits, but, as a result of selectivity, intellectual property and other issues, the projects do not result in lead declarations. In this article, HTS success is defined as the fraction of HTS campaigns that advance into the later stages of drug discovery, and the major influencing factors are examined. Interestingly, screening compounds in individual wells or in mixtures did not have a major impact on the HTS success and, equally interesting, there was no difference in the progression rates of biochemical and cell-based assays. Particular target types, assay technologies, structure-activity relationships and powder availability had a much greater impact on success as defined above. In addition, significant mutual dependencies can be observed - while one assay format works well with one target type, this situation might be completely reversed for a combination of the same readout technology with a different target type. The results and opinions presented here should be regarded as groundwork, and a plethora of factors that influence the fate of a project, such as biophysical measurements, chemical attractiveness of the hits, strategic reasons and safety pharmacology, are not covered here. Nonetheless, it is hoped that this information will be used industry-wide to improve success rates in terms of hits progressing into exploratory chemistry and beyond. The support that can be obtained from new in silico approaches to phase transitions are also described, along with the gaps they are designed to fill.

Maximising use of in vitro ADMET tools to predict in vivo bioavailability and safety.

The drastic increase in the costs for discovering and developing a new drug and the high attrition rate of development candidates led to shifting of drug discovery strategy to parallel assessment of comprehensive drug properties along with efficacy. The article reviews the benefits and caveats of implementing comprehensive in vitro tools in early drug discovery and their impact on addressing in vivo ADMET issues. With the proposal of four-barrier profiling paradigm and employment of integrated risk assessment, one can exponentially enhance the predictive power of those in vitro tools by taking into consideration the interplays among those profiling parameters. An 'Exposure Cube' is proposed to promote collective employment of solubility/dissolution, permeability and metabolic clearance to address in vivo exposure and to direct optimization of new chemical entities in drug discovery.

High-throughput in vitro profiling assays: lessons learnt from experiences at Novartis.

This article reviews the use of a selection of in vitro assays implemented at Novartis and intends to address exposure and safety in early drug discovery. The authors' own experience, based on a large number of 'real' drug discovery compounds, is described to reflect on what has worked, where improvement is needed and how to best use the data for decision making. Possible strategies are discussed, and guidelines are provided on how to organise assays, extract value and contribute knowledge from the data.

Keynote review: in vitro safety pharmacology profiling: an essential tool for successful drug development.

Broad-scale in vitro pharmacology profiling of new chemical entities during early phases of drug discovery has recently become an essential tool to predict clinical adverse effects. Modern, relatively inexpensive assay technologies and rapidly expanding knowledge about G-protein coupled receptors, nuclear receptors, ion channels and enzymes have made it possible to implement a large number of assays addressing possible clinical liabilities. Together with other in vitro assays focusing on toxicology and bioavailability, they provide a powerful tool to aid drug development. In this article, we review the development of this tool for drug discovery, its appropriate use and predictive value.

A novel membrane potential-sensitive fluorescent dye improves cell-based assays for ion channels.

The study of ion channel-mediated changes in membrane potential using the conventional bisoxonol fluorescent dye DiBAC(4)(3) has several limitations, including a slow onset of response and multistep preparation, that limit both the fidelity of the results and the throughput of membrane potential assays. Here, we report the characterization of the FLIPR Membrane Potential Assay Kit (FMP) in cells expressing voltage- and ligand-gated ion channels. The steady-state and kinetics fluorescence properties of FMP were compared with those of DiBAC(4)(3), using both FLIPR and whole-cell patch-clamp recording. Our experiments with the voltage-gated K(+) channel, hElk-1, revealed that FMP was 14-fold faster than DiBAC(4)(3) in response to depolarization. On addition of 60 mM KCl, the kinetics of fluorescence changes of FMP using FLIPR were identical to those observed in the electrophysiological studies using whole-cell current clamp. In addition, KCl concentration-dependent increases in FMP fluorescence correlated with the changes of membrane potential recorded in whole-cell patch clamp. In studies examining vanilloid receptor-1, a ligand-gated nonselective cation channel, FMP was superior to DiBAC(4)(3) with respect to both kinetics and amplitude of capsaicin-induced fluorescence changes. FMP has also been used to measure the activation of K(ATP) and hERG. Thus this novel membrane potential dye represents a powerful tool for developing high-throughput screening assays for ion channels.