G-protein coupled receptor assays: to measure affinity or efficacy that is the question

Cell-based assays have always played an important role in the pharmaceutical industry, providing information about the functional effects of compounds. These functional assays have traditionally accompanied facile biochemical high throughput screening programmes, being applied as secondary assays in the later stages of lead development. However, with the disappointing reality that there is not likely to be a plethora of novel, druggable targets in the post-genomic era, the role of cell-based assays in drug discovery is beginning to change. Competition to develop the "best" agents for well established targets and find more effective ways of identifying "novel" agents is driving the industry towards a "quality" versus "quantity" approach. Advances in genetic engineering, automation compatible functional assay technologies and the introduction of more sophisticated robotic systems, have facilitated the application of cell-based assays to primary screening. However, despite some apparent success to move these assays into the routine "toolbox" for high throughput screening, certain preconceptions and concerns about cell-based assays persist and the subject remains a topic of much debate. Here we use examples from the screening portfolio at Pfizer, Sandwich, to discuss the practical and theoretical considerations of employing cell-based assays in HTS with a focus on G-protein coupled receptors.     

Whole cell strategies based on lux genes for high throughput applications toward new antimicrobials

The discovery/development of novel drug candidates has witnessed dramatic changes over the last two decades. Old methods to identify lead compounds are not suitable to screen wide libraries generated by combinatorial chemistry techniques. High throughput screening (HTS) has become irreplaceable and hundreds of different approaches have been described. Assays based on purified components are flanked by whole cell-based assays, in which reporter genes are used to monitor, directly or indirectly, the influence of a chemical over the metabolism of living cells. The most convenient and widely used reporters for real-time measurements are luciferases, light emitting enzymes from evolutionarily distant organisms. Autofluorescent proteins have been also extensively employed, but proved to be more suitable for end-point measurements, in situ applications - such as the localization of fusion proteins in specific subcellular compartments - or environmental studies on microbial populations. The trend toward miniaturization and the technical advances in detection and liquid handling systems will allow to reach an ultra high throughput screening (uHTS), with 100,000 of compounds routinely screened each day. Here we show how similar approaches may be applied also to the search for new and potent antimicrobial agents.     

Micro-patterned porous substrates for cell-based assays

In the search for new therapeutic chemicals, lab-on-a-chip systems have recently emerged as innovative and efficient tools for cell-based assays and high throughput screening. Here, we describe a novel, versatile and simple device for cell-based assays at the bench-top. We created spatial variations of porosity on the surface of a membrane filter by microcontact printing with a biocompatible polymer (PDMS). We called such systems Micro-Printed Membranes (μPM). Active compounds dispensed on the porous areas, where the membrane pores are not clogged by the polymer, can cross the membrane and reach cells growing on the opposite side. Only cells immediately below those porous areas could be stimulated by chemicals. We performed proof-of-principle experiments using Hoechst nuclear staining, calcein-AM cell viability assay and destabilization of the cytoskeleton organisation by cytochalasin B. Resulting fluorescent staining properly matched the drops positioning and no cross-contaminations were observed between adjacent tests. This well-less cell-based screening system is highly flexible by design and it enables multiple compounds to be tested on the same cell tissue. Only low sample volumes in the microlitre range are required. Moreover, chemicals can be delivered sequentially and removed at any time while cells can be monitored in real time. This allows the design of complex, sequential and combinatorial drug assays. μPMs appear as ideal systems for cell-based assays. We anticipate that this lab-on-chip device will be adapted for both manual and automated high content screening experiments.     

Label-free cell-based functional assays

Label-free technologies based on electrical impedance or refractive index are new tools for measuring a cell-based functional response. Although the technologies are relatively new to high throughput screening cell-based applications, they are rapidly generating interest in that they are able to measure a phenotypic response using cells natively expressing the target protein without using dyes or cellular extracts. In addition, one can measure the cellular response using a kinetic mode resulting in an assay potentially rich in content. This article will describe these technologies and their applications in measuring cell proliferation, cell attachment and spreading, cell apoptosis and their application for several receptor target classes, including receptor tyrosine kinases and G protein-coupled receptors. The potential utility and drawbacks of these tools for high throughput screening, directed screening and compound profiling will also be discussed.     

A miniaturized glucocorticoid receptor translocation assay using enzymatic fragment complementation evaluated with qHTS

Nuclear translocation is an important step in glucocorticoid receptor (GR) signaling and assays that measure this process allow the identification of nuclear receptor ligands independent of subsequent functional effects. To facilitate the identification of GR-translocation agonists, an enzyme fragment complementation (EFC) cell-based assay was scaled to a 1536-well plate format to evaluate 9,920 compounds using a quantitative high throughput screening (qHTS) strategy where compounds are assayed at multiple concentrations. In contrast to conventional assays of nuclear translocation the qHTS assay described here was enabled on a standard luminescence microplate reader precluding the requirement for imaging methods. The assay uses beta-galactosidase alpha complementation to indirectly detect GR-translocation in CHO-K1 cells. 1536-well assay miniaturization included the elimination of a media aspiration step, and the optimized assay displayed a Z' of 0.55. qHTS yielded EC(50) values for all 9,920 compounds and allowed us to retrospectively examine the dataset as a single concentration-based screen to estimate the number of false positives and negatives at typical activity thresholds. For example, at a 9 microM screening concentration, the assay showed an accuracy that is comparable to typical cell-based assays as judged by the occurrence of false positives that we determined to be 1.3% or 0.3%, for a 3sigma or 6sigma threshold, respectively. This corresponds to a confirmation rate of approximately 30% or approximately 50%, respectively. The assay was consistent with glucocorticoid pharmacology as scaffolds with close similarity to dexamethasone were identified as active, while, for example, steroids that act as ligands to other nuclear receptors such as the estrogen receptor were found to be inactive.     

A high throughput glucocerebrosidase assay using the natural substrate glucosylceramide

Glucocerebrosidase is a lysosomal enzyme that catalyzes the hydrolysis of glucosylceramide to form ceramide and glucose. A deficiency of lysosomal glucocerebrosidase due to genetic mutations results in Gaucher disease, in which glucosylceramide accumulates in the lysosomes of certain cell types. Although enzyme replacement therapy is currently available for the treatment of type 1 Gaucher disease, the neuronopathic forms of Gaucher disease are still not treatable. Small molecule drugs that can penetrate the blood-brain barrier, such as pharmacological chaperones and enzyme activators, are new therapeutic approaches for Gaucher disease. Enzyme assays for glucocerebrosidase are used to screen compound libraries to identify new lead compounds for drug development for the treatment of Gaucher disease. But the current assays use artificial substrates that are not physiologically relevant. We developed a glucocerebrosidase assay using the natural substrate glucosylceramide coupled to an Amplex-red enzyme reporting system. This assay is in a homogenous assay format and has been miniaturized in a 1,536-well plate format for high throughput screening. The assay sensitivity and robustness is similar to those seen with other glucocerebrosidase fluorescence assays. Therefore, this new glucocerebrosidase assay is an alternative approach for high throughput screening.     

Development of a novel high-throughput assay for the investigation of GlyT-1b neurotransmitter transporter function

The glycine transporter (GlyT-1b) is a Na(+)/Cl(-)-dependent electrogenic transporter which mediates the rapid re-uptake of glycine from the synaptic cleft. Based on its tissue distribution, GlyT-1 has been suggested to co-localise with the NMDA receptor where it may modulate the concentration of glycine at its co-agonist binding site. This data has led to GlyT-1 inhibitors being proposed as targets for disorders such as schizophrenia and cognitive dysfunction. Radiolabelled uptake assays (e.g. [(3)H]glycine) have been traditionally used in compound screening to identify glycine transporter inhibitors. While such an assay format is useful for testing limited numbers of compounds, the identification of novel glycine uptake inhibitors requires a functional assay compatible with high-throughput screening (HTS) of large compound libraries. Here, the authors present the development of a novel homogenous cell-based assay using the FLIPR membrane potential blue dye (Molecular Devices) and FLEXstation. Pharmacological data for the GlyT-1 inhibitors Org 24598 and ALX 5407 obtained using this novel electrogenic assay correlated well with the conventional [(3)H]-glycine uptake assay format. Furthermore, the assay has been successfully miniaturised using FLIPR(3) and therefore has the potential to be used for high-throughput screening.     

Fluorescence polarization is a useful technology for reagent reduction in assay miniaturization

The use of fluorescence polarization (FP) has increased significantly in the development of sensitive and robust assays for high throughput screening of chemical compound libraries during the past few years. In this study, we show that FP is a useful assay miniaturization technology for reagent reduction during high throughput screening. We developed and optimized several FP assays for binding to estrogen receptor alpha and two protein kinases with an assay volume of 100 microl. Without any re-optimization, a consistent signal window was maintained in 384- or 1536-well format when the assay volume varied from 2.5-100 microl at constant concentrations of all assay components. In contrast, the signal window decreased with decreasing assay volume at constant reagent concentration in the protein kinase C scintillation proximity assay (SPA) and prompt fluorescence assay. In addition, the effect of evaporation on the signal window was minimal for the FP assays. Our study suggests that FP is superior to SPA and prompt fluorescence in terms of reagent reduction in the miniaturized assay format.     

Dioxin analysis in feed: cell-based assay versus mass spectrometry method

In the determination of contaminants (dioxins, polychlorinated biphenyls, polyaromatic hydrocarbons), cell-based assays are useful methods for screening purposes: they are mainly characterized by high sample throughput and lower costs than the Mass Spectrometry (MS)-based methods. Although cell-based assays can be sensitive enough for the determination of dioxins and related substances in agreement with the presently tolerable limits in food and feed (Regulation No. 2375/2001/EC and Directive 2003/57/EC respectively), their lack of specificity make their use rather questionable in control laboratories. In this paper, we present and compare results obtained from the analysis of a limited number of feed samples by both gas chromatography-high resolution mass spectrometry (GC-HRMS) and cell-based assay (DR-CALUX: dioxin responsive-chemically activated luciferase gene expression) methods. The DR-CALUX screening led to less than 10% false non-compliant and no false compliant results. In addition, there is a good correlation between GC-HRMS and DR-CALUX data. However, these preliminary results have to be confirmed on a larger number of samples to demonstrate that total toxic equivalent (TEQ), including dioxins, furans and dioxin-like polychlorobiphenyls (PCBs) can be monitored in feed and food with a cell-based assay. Presented at AOAC Europe/Eurachem Symposium March 2005, Brussels, Belgium     

临近闪烁分析法在高通量筛选中的应用

起源于放射性免疫分析的临近闪烁分析法(scintillation proximity assay,SPA)是一种均相、灵敏、快速和简便的基于闪烁载体的分析平台。该平台可用于筛选药物靶点的先导化合物和研究其生理过程。由于无需分离,易于固定药物靶点和检测其活性,SPA成为一种重要的高通量筛选方法。由于放射性标记分子和亲和标签分子的多样化和商业化、以及液闪计数器和液相操作等技术的发展,SPA已经广泛用于受体结合、高通量药物筛选、酶分析、放射性免疫分析、蛋白质-蛋白质相互作用和细胞水平分析等方面。本文阐述了SPA原理,讨论了其关键技术(包括闪烁载体、液闪计数器和放射性标记分子),分析了其评价体系;同时简述了SPA分析的发展, 并介绍了其在高通量筛选中的应用实例, 归纳了存在的问题,给出了未来的发展趋势。目前,基于SPA和荧光分析方法已成为高通量药物筛选的热点研究领域, 这些筛选技术的革新必然提升我们对细胞体系生物学的全面理解和促进先导化合物筛选过程的显著进步。     

Cassette-accelerated rapid rat screen: a systematic procedure for the dosing and liquid chromatography/atmospheric pressure ionization tandem mass spectrometric analysis of new chemical entities as part of new drug discovery

This report addresses the continuing need for increased throughput in the evaluation of new chemical entities (NCEs) in terms of their pharmacokinetic (PK) parameters by describing an alternative procedure for increasing the throughput of the in vivo screening of NCEs in the oral rat PK model. The new approach is called "cassette-accelerated rapid rat screen" (CARRS). In this assay, NCEs are dosed individually (n = 2 rats/compound) in batches of six compounds per set. The assay makes use of a semi-automated protein precipitation procedure for sample preparation in a 96-well plate format. The liquid chromatography/atmospheric pressure ionization tandem mass spectrometry (LC/API-MS/MS) assay is also streamlined by analyzing the samples as "cassettes of six". Using this new approach, a threefold increase in throughput was achieved over the previously reported "rapid rat screen".     

Automated high content screening for phosphoinositide 3 kinase inhibition using an AKT 1 redistribution assay

High Content Screening (HCS), a combination of fluorescence microscopic imaging and automated image analysis, has become a frequently applied tool to study test compound effects in cellular disease-modelling systems. In this work, we established a medium to high throughput HCS assay in the 384-well format to measure cellular type I phosphoinositide 3 kinase (PI3K) activity. Type I PI3K is involved in several intracellular pathways such as cell survival, growth and differentiation as well as immunological responses. As a cellular model system we used Chinese Hamster Ovary (CHO) cells that had been stably transfected with human insulin receptor (hIR) and an AKT1-enhanced green fluorescent protein (EGFP) fusion construct. Upon stimulation of the hIR with insulin-like growth factor-1 (IGF-1), PI3K was activated to phosphorylate phosphatidylinositol (PtdIns)-4,5-bisphosphate at the 3-position, resulting in the recruitment of AKT1-EGFP to the plasma membrane. The AKT1-EGFP redistribution assay was robust and displayed little day-to-day variability, the quantification of the fluorescence intensity associated with plasma membrane spots delivered good Z' statistics. A novel format of compound dose-response testing was employed using serial dilutions of test compounds across consecutive microtiter plates (MTPs). The dose response testing of a PI3K inhibitor series provided reproducible IC50 values. The profiling of the redistribution assay with isoform-selective inhibitors indicates that PI3Kalpha is the main isoform activated in the CHO host cells after IGF-1 stimulation. Toxic compound side effects could be determined using automated image analysis. We conclude that the AKT1-EGFP redistribution assay represents a solid medium/high throughput screening (MTS/HTS) format to determine the cellular activity of PI3K inhibitors under conditions of growth factor stimulation.     

Droplet-based microfluidic platforms for the encapsulation and screening of Mammalian cells and multicellular organisms

High-throughput, cell-based assays require small sample volumes to reduce assay costs and to allow for rapid sample manipulation. However, further miniaturization of conventional microtiter plate technology is problematic due to evaporation and capillary action. To overcome these limitations, we describe droplet-based microfluidic platforms in which cells are grown in aqueous microcompartments separated by an inert perfluorocarbon carrier oil. Synthesis of biocompatible surfactants and identification of gas-permeable storage systems allowed human cells, and even a multicellular organism (C. elegans), to survive and proliferate within the microcompartments for several days. Microcompartments containing single cells could be reinjected into a microfluidic device after incubation to measure expression of a reporter gene. This should open the way for high-throughput, cell-based screening that can use >1000-fold smaller assay volumes and has approximately 500x higher throughput than conventional microtiter plate assays.     

MEMS microwell and microcolumn arrays: novel methods for high-throughput cell-based assays

Although the cell-based assay is becoming more popular for high throughput drug screening and the functional characterization of disease-associated genes, most researchers in these areas do not use it because it is a complex and expensive process. We wanted to create a simple method of performing an on-chip cell-based assay. To do this, we used micro-electro-mechanical systems (MEMS) to fabricate a microwell array chip comprised of a glass substrate covered with a photoresist film patterned to form multiple microwells and tested it in two reverse transfection experiments, an exogenous gene expression study and an endogenous gene knockdown study. It was used effectively in both. Then, using the same MEMS technology, we fabricated a complementary microcolumn array to be used as a drug carrier device to topically apply drugs to cells cultured in the microwell array. We tested the effectiveness of microwell-microcolumn on-chip cell-based assay by using it in experiments to identify epidermal growth factor receptor (EGFR) activity inhibitors, for which it was found to provide effective high throughput and high content functional screening. In conclusion, this new method of cell-based screening proved to be a simple and efficient method of characterizing gene function and discovering drug leads.     

Label free binding assay with spectroscopic detection for pharmaceutical screening

Many therapeutic drugs exert their effects by interaction with well defined molecular targets. Increasing knowledge in molecular biology allows identification of more and more molecular key compounds and in consequence a molecular approach to disease and therapy. As binding of drugs to their target compounds is a key event, binding assays with an appropriate target molecule are useful means for primary screening of novel substances. We have investigated the potential of thin film interference spectroscopy (RIFS) as a label free detection method for pharmaceutical screening in a binding inhibition assay. To meet the throughput requirements in pharmaceutical screening a parallel detection system based on imaging spectroscopy was constructed. Thrombin/thrombin inhibitor interaction was investigated as a model system. The thin film transducer was covalently modified with a thrombin inhibitor. Specific binding of thrombin and binding inhibition by inhibitor compounds could be observed. A test cycle of less than 10 min could be reached. The parallel setup allows the simultaneous detection of 96 binding curves and can reach a throughput of more than 10⁶ samples per year.     

High-throughput screening of small molecules in miniaturized mammalian cell-based assays involving post-translational modifications

BACKGROUND: Fully adapting a forward genetic approach to mammalian systems requires efficient methods to alter systematically gene products without prior knowledge of gene sequences, while allowing for the subsequent characterization of these alterations. Ideally, these methods would also allow function to be altered in a temporally controlled manner. RESULTS: We report the development of a miniaturized cell-based assay format that enables a genetic-like approach to understanding cellular pathways in mammalian systems using small molecules, rather than mutations, as the source of gene-product alterations. This whole-cell immunodetection assay can sensitively detect changes in specific cellular macromolecules in high-density arrays of mammalian cells. Furthermore, it is compatible with screening large numbers of small molecules in nanoliter to microliter culture volumes. We refer to this assay format as a 'cytoblot', and demonstrate the use of cytoblotting to monitor biosynthetic processes such as DNA synthesis, and post-translational processes such as acetylation and phosphorylation. Finally, we demonstrate the applicability of these assays to natural-product screening through the identification of marine sponge extracts exhibiting genotype-specific inhibition of 5-bromodeoxyuridine incorporation and suppression of the anti-proliferative effect of rapamycin. CONCLUSIONS: We show that cytoblots can be used for high-throughput screening of small molecules in cell-based assays. Together with small-molecule libraries, the cytoblot assay can be used to perform chemical genetic screens analogous to those used in classical genetics and thus should be applicable to understanding a wide variety of cellular processes, especially those involving post-transitional modifications.     

Application and utilization of chemoinformatics tools in lead generation and optimization

The process of Drug Discovery is a complex and high risk endeavor that requires focused attention on experimental hypotheses, the application of diverse sets of technologies and data to facilitate high quality decision-making. All is aimed at enhancing the quality of the chemical development candidate(s) through clinical evaluation and into the market. In support of the lead generation and optimization phases of this endeavor, high throughput technologies such as combinatorial/high throughput synthesis and high throughput and ultra-high throughput screening, have allowed the rapid analysis and generation of large number of compounds and data. Today, for every analog synthesized 100 or more data points can be collected and captured in various centralized databases. The analysis of thousands of compounds can very quickly become a daunting task. In this article we present the process we have developed for both analyzing and prioritizing large sets of data starting from diversity and focused uHTS in support of lead generation and secondary screens supporting lead optimization. We will describe how we use informatics and computational chemistry to focus our efforts on asking relevant questions about the desired attributes of a specific library, and subsequently in guiding the generation of more information-rich sets of analogs in support of both processes.     

Application of affinity selection-mass spectrometry assays to purification and affinity-based screening of the chemokine receptor CXCR4

Affinity selection-mass spectrometry (AS-MS) is a sensitive technology for identifying small molecules that bind to target proteins, and assays enabled by AS-MS can be used to delineate relative binding affinities of ligands for proteins. 'Indirect' AS-MS assays employ size-exclusion techniques to separate target-ligand complexes from unbound ligands, and target-associated ligands are then specifically detected by liquid chromatography mass spectrometry. We report how indirect AS-MS binding assays with known reference control compounds were used as guideposts for development of an optimized purification method for CXCR4, a G-protein coupled chemokine receptor, for which we sought novel antagonists. The CXCR4 purification method that was developed was amenable to scale-up and enabled the screening of purified recombinant human CXCR4 against a large combinatorial library of small molecules by high throughput indirect AS-MS. The screen resulted in the discovery of new ligands that competed off binding of reference compounds to CXCR4 in AS-MS binding assays and that antagonized SDF1α-triggered responses and CXCR4-mediated HIV1 viral uptake in cell-based assays. This report provides a methodological paradigm whereby indirect AS-MS-based ligand binding assays may be used to guide optimal integral membrane protein purification methods that enable downstream affinity selection-based applications such as high throughput AS-MS screens.     

Advanced dynamic monitoring of cellular status using label-free and non-invasive cell-based sensing technology for the prediction of anticancer drug efficacy

Quantitative evaluation of anticancer drug efficacy using in vitro cell-based assays is useful for cancer patients, particularly those who show unconventional cancer development. Nevertheless, conventional chemosensitivity testing often requires widely used labeling agents and time-consuming laboratory procedures that provide low reliability. Label-free non-invasive cell-based assays are desired for dynamic monitoring of cellular status. This critical review first describes conventional chemosensitivity testing and then advanced label-free cell-based technology used to screen anticancer drugs through dynamic monitoring of cellular status, focusing on dosage and the use of drug-resistant cancer cells. Results from label-free cell-based approaches are compared with those of conventional chemosensitivity testing. The cellular statuses, addressed in terms of respective mechanisms and disadvantages, are extracellular fluxes of proton (H⁺), O₂, and anticancer drugs, cell morphology changes, cell–environment interaction, and mitochondrial membrane potential. Finally, a cell-based systems outlook is presented. This paper represents a step toward efficient and accurate initial screening of anticancer drugs and development of compounds and their combined use to achieve pharmacodynamic and pharmacokinetic interactions, and chemotherapy evaluation of particular anticancer drugs for individual patients.