Miniaturization of intracellular calcium functional assays to 1536-well plate format using a fluorometric imaging plate reader

The measurement of intracellular calcium response transients in living mammalian cells is a popular functional assay for identification of agonists and antagonists to receptors or channels of pharmacological interest. In recent years, advances in fluorescence-based detection techniques and automation technologies have facilitated the adaptation of this assay to 384-well microplate format high-throughput screening (HTS) assays. However, the cost and time required performing the intracellular calcium HTS assays in the 384-well format can be prohibitive for HTS campaigns of greater than 1 x 10(6) wells. For these reasons, it is attractive to miniaturize intracellular calcium functional assays to the 1536-well microplate format, where assay volumes and plate throughput can be decreased by several fold. The focus of the research described in this article is the miniaturization of an intracellular calcium assay to 1536-well plate format. This was accomplished by modifying the hardware and software of a fluorometric imaging plate reader (FLIPR) to enable transfer of nanoliters of test compound directly to a 1536-well assay plate, and measure the resulting calcium response from all 1536 wells simultaneously. An intracellular calcium functional assay against the rat muscarinic acetylcholine receptor subtype 1 (rmAchR1) G-protein coupled receptor (GPCR) was miniaturized and executed on this modified instrument. In experiments measuring the activity of known muscarinic receptor agonists and antagonists, the miniaturized FLIPR assay gave EC(50) and IC(50) values and rank order potency comparable to the 384-well format assays. Calculated Z' factors for the miniaturized agonist and antagonist assays were, respectively, 0.56 +/- 0.21 and 0.53 +/- 0.22, which were slightly higher (Z'(agonist) = 0.55 +/- 0.33) and lower (Z'(antagonist) = 0.70 +/- 0.18) than the corresponding values in the 384-well assays. A mock agonist HTS campaign against the muscarinic receptor in miniaturized format was able to identify all wells spiked with the rmAchR1 agonist carbachol.     

Miniaturization of fluorescence polarization receptor-binding assays using CyDye-labeled ligands

Fluorescence polarization (FP) is an established technique for the study of biological interactions and is frequently used in the high-throughput screening (HTS) of potential new drug targets. This work describes the miniaturization of FP receptor assays to 1536-well formats for use in HTS. The FP assays were initially developed in 384-well microplates using CyDye-labeled nonpeptide and peptide ligands. Receptor expression levels varied from approximately 1 to 10 pmols receptor per mg protein, and ligand concentrations were in the 0.5- to 1.0-nM range. The FP assays were successfully miniaturized to 1536-well formats using Cy3B-labeled ligands, significantly reducing reagent consumption, particularly the receptor source, without compromising assay reliability. Z' factor values determined for the FP receptor assays in both 384- and 1536-well formats were found to be > 0.5, indicating the assays to be robust, reliable, and suitable for HTS purposes.     

Comparison of miniaturized time-resolved fluorescence resonance energy transfer and enzyme-coupled luciferase high-throughput screening assays to discover inhibitors of Rho-kinase II (ROCK-II)

Kinases are important drug discovery targets for a wide variety of therapeutic indications; consequently, the measurement of kinase activity remains a common high-throughput screening (HTS) application. Recently, enzyme-coupled luciferase-kinase (LK) format assays have been introduced. This format measures luminescence resulting from metabolism of adenosine triphosphate (ATP) via a luciferin/luciferase-coupled reaction. In the research presented here, 1536-well format time-resolved fluorescence resonance energy transfer (TR-FRET) and LK assays were created to identify novel Rho-associated kinase II (ROCK-II) inhibitors. HTS campaigns for both assays were conducted in this miniaturized format. It was found that both assays were able to consistently reproduce the expected pharmacology of inhibitors known to be specific to ROCK-II (fasudil IC50: 283 +/- 27 nM and 336 +/- 54 nM for TR-FRET and LK assays, respectively; Y-27632 IC50: 133 +/- 7.8 nM and 150 +/- 22 nM for TR-FRET and LK assays, respectively). In addition, both assays proved robust for HTS efforts, demonstrating excellent plate Z' values during the HTS campaign (0.84 +/- 0.03; 0.72 +/- 0.05 for LK and TR-FRET campaigns, respectively). Both formats identified scaffolds of known and novel ROCK-II inhibitors with similar sensitivity. A comparison of the performance of these 2 assay formats in an HTS campaign was enabled by the existence of a subset of 25,000 compounds found in both our institutional and the Molecular Library Screening Center Network screening files. Analysis of the HTS campaign results based on this subset of common compounds showed that both formats had comparable total hit rates, hit distributions, amount of hit clusters, and format-specific artifact. It can be concluded that both assay formats are suitable for the discovery of ROCK-II inhibitors, and the choice of assay format depends on reagents and/or screening technology available.     

Using ligand-induced conformational change to screen for compounds targeting G-protein-coupled receptors

The authors describe a novel drug strategy designed as a primary screen to discover either antagonist or agonist compounds targeting G-protein-coupled receptors (GPCRs). The incorporation of a nuclear localization sequence (NLS, a 5 amino acid substitution), in a location in helix 8 of the GPCR structure, resulted in ligand-independent receptor translocation from the cell surface to the nucleus. Blockade of the GPCR-NLS translocation from the cell surface was achieved by either antagonist or agonist treatments, each achieving their result in a sensitive concentration-dependent manner. GPCR-NLS translocation and blockade occurred regardless of the identity of the G-protein-coupling, and thus this assay is also ideally suited for identification of compounds targeting orphan GPCRs. The GPCR-NLS trafficking was visualized by fusion to fluorescent detectable proteins. Quantification of this effect was measured by determining the density of cell surface receptors, using enzyme fragment complementation in a manner suitable for high-throughput screening. Thus, the authors have developed a cellular assay for GPCRs suitable for compound screening without requiring prior identification of an agonist or knowledge of G-protein-coupling.     

Characterization of the 5-HT2b receptor in evaluation of aequorin detection of calcium mobilization for miniaturized GPCR high-throughput screening

Fluorescent detection of calcium mobilization has been used successfully to identify modulators of G-protein-coupled receptors (GPCRs); however, inherent issues with fluorescence may limit its potential for high-throughput screening miniaturization. The data presented here demonstrate that the calcium-sensitive photoprotein aequorin (AequoScreen), when compared with FLUO-4 in the same cellular background, allows for miniaturization of functional kinetic calcium flux assays, in which the rank order of potency and efficacy was maintained for a series of diverse small-molecule modulators. Small-volume (<10 microL) 384- and 1536-well aequorin assays were implemented by integration of acoustic dispensing (Echo 550) and kinetic flash luminometry (CyBi Lumax). The enhanced high signal-to-background ratios observed relative to fluorescence were readily manipulated by altering per-well cell densities and yielded acceptable screening statistics in miniaturized format for both agonist and antagonist screening scenarios. In addition, the authors demonstrate the feasibility of using agonist concentrations less than EC(50) in a miniaturized antagonist assay. These features, coupled with improved sample handling, should enhance sensitivity and provide the benefits of miniaturization including cost reduction and throughput gains.     

Digital Imaging as a Detection Method for a Fluorescent Protease Assay in 96-Well and Miniaturized Assay Plate Formats

The demand to increase throughput in HTS programs, without a concomitant addition to costs, has grown significantly during the past few years. One approach to handle this demand is assay miniaturization, which can provide greater throughput, as well as significant cost savings through reduced reagent costs. Currently, one of the major challenges facing assay miniaturization is the ability to detect the assay signal accurately and rapidly in miniaturized formats. Digital imaging is a detection method that can measure fluorescent or luminescent signals in these miniaturized formats. In this study, an imaging system capable of detecting the signal from a fluorescent protease assay in multiple plate formats was used to evaluate this detection method in an HTS environment. A direct comparison was made between the results obtained from the imaging system and a fluorescent plate reader by screening 8,800 compounds in a 96-well plate format. The imaging system generated similar changes in relative signal for each well in the screen, identified the same active compounds, and yielded similar IC(50) values as compared to the plate reader. When a standard protease inhibitor was evaluated in 96-, 384-, 864-, and 1536-well plates using imaging detection, similar IC(50) values were obtained. Furthermore, similar dose-response curves were generated for the compound in 96- and 384-well assay plates read in a plate reader. These results provide support for digital imaging as an accurate and rapid detection method for high-density microtiter plates.     

An Ultra High-Throughput,Whole-Animal Screen for Small Molecule Modulators of a Specific Genetic Pathway in Caenorhabditis elegans

High-throughput screening (HTS) is a powerful approach to drug discovery, but many lead compounds are found to be unsuitable for use in vivo after initial screening. Screening in small animals like C. elegans can help avoid these problems, but this system has been limited to screens with low-throughput or no specific molecular target. We report the first in vivo 1536-well plate assay for a specific genetic pathway in C. elegans. Our assay measures induction of a gene regulated by SKN-1, a master regulator of detoxification genes. SKN-1 inhibitors will be used to study and potentially reverse multidrug resistance in parasitic nematodes. Screens of two small commercial libraries and the full Molecular Libraries Small Molecule Repository (MLSMR) of ∼364,000 compounds validate our platform for ultra HTS. Our platform overcomes current limitations of many whole-animal screens and can be widely adopted for other inducible genetic pathways in nematodes and humans.     

Use of fluorescence polarization detection for the measurement of fluopeptidetm binding to G protein-coupled receptors

G protein-coupled receptors (GPCRs) represent the single largest molecular target of therapeutic drugs currently on the market, and are also the most common target in high throughput screening assays designed to identify potential new drug candidates. A large percentage of these assays are now formatted as radioligand binding assays. Fluorescence polarization ligand binding assays can offer a non-rad alternative to radioligand binding assays. In addition, fluorescence polarization assays are a homogenous format that is easy to automate for high throughput screening. We have developed a series of peptide ligands labeled with the fluorescent dye BODIPY TMR whose binding to GPCRs can be detected using fluorescence polarization methodology. BODIPY TMR has advantages over the more commonly used fluorescein dye in high throughput screening (HTS) assays due to the fact that its excitation and emission spectra are red-shifted approximately 50 nm relative to fluorescein. Assays based on BODIPY TMR ligands are therefore less susceptible to interference from tissue auto-fluorescence in the assay matrix, or the effects of colored or fluorescent compounds in the screening libraries. A series of BODIPY TMR labeled peptides have been prepared that bind to a range of GPCRs including melanin concentrating hormone, bradykinin, and melanocortin receptors. Conditions have been optimized in order to utilize a comparable amount of receptor membrane preparation as is used in a radioligand binding assay. The assays are formatted in 384-well microplates with a standard volume of 40 microL. We have compared the assays across the different fluorescence polarization (FP) readers available to determine the parameters for each instrument necessary to achieve the required precision.     

Highly miniaturized formats for in vitro drug metabolism assays using vivid fluorescent substrates and recombinant human cytochrome P450 enzymes

Highly miniaturized P450 screening assays designed to enable facile analysis of P450 drug interactions in a 1536-well plate format with the principal human cytochrome P450 enzymes (CYP3A4, 2D6, 2C9, 2C19, and 1A2) and Vivid fluorogenic substrates were developed. The detailed characterization of the assays included stability, homogeneity, and reproducibility of the recombinant P450 enzymes and the kinetic parameters of their reactions with Vivid fluorogenic substrates, with a focus on the specific characteristics of each component that enable screening in a low-volume 1536-well plate assay format. The screening assays were applied for the assessment of individual cytochrome P450 inhibition profiles with a panel of selected assay modifiers, including isozyme-specific substrates and inhibitors. IC(50) values obtained for the modifiers in 96- and 1536-well plate formats were similar and comparable with values obtained in assays with conventional substrates. An overall examination of the 1536-well assay statistics, such as signal-to-background ratio and Z' factor, demonstrated that these assays are a robust, successful, and reliable tool to screen for cytochrome P450 metabolism and inhibition in an ultra-high-throughput screening format.     

Adaptation of aequorin functional assay to high throughput screening

AequoScreen, a cellular aequorin-based functional assay, has been optimized for luminescent high-throughput screening (HTS) of G protein-coupled receptor (GPCRs). AequoScreen is a homogeneous assay in which the cells are loaded with the apoaequorin cofactor coelenterazine, diluted in assay buffer, and injected into plates containing the samples to be tested. A flash of light is emitted following the calcium increase resulting from the activation of the GPCR by the sample. Here we have validated a new plate reader, the Hamamatsu Photonics FDSS6000, for HTS in 96- and 384-well plates with CHO-K1 cells stably coexpressing mitochondrial apoaequorin and different GPCRs (AequoScreen cell lines). The acquisition time, plate type, and cell number per well have been optimized to obtain concentration-response curves with 4000 cells/well in 384-well plates and a high signal:background ratio. The FDSS6000 and AequoScreen cell lines allow reading of twenty 96- or 384-well plates in 1 h with Z' values of 0.71 and 0.78, respectively. These results bring new insights to functional assays, and therefore reinforce the interest in aequorin-based assays in a HTS environment.     

USE OF FLUORESCENCE POLARIZATION DETECTION FOR THE MEASUREMENT OF FLUOPEPTIDE™ BINDING TO G PROTEIN-COUPLED RECEPTORS

ABSTRACT

G protein-coupled receptors (GPCRs) represent the single largest molecular target of therapeutic drugs currently on the market, and are also the most common target in high throughput screening assays designed to identify potential new drug candidates. A large percentage of these assays are now formatted as radioligand binding assays. Fluorescence polarization ligand binding assays can offer a non-rad alternative to radioligand binding assays. In addition, fluorescence polarization assays are a homogenous format that is easy to automate for high throughput screening. We have developed a series of peptide ligands labeled with the fluorescent dye BODIPY® TMR whose binding to GPCRs can be detected using fluorescence polarization methodology. BODIPY® TMR has advantages over the more commonly used fluorescein dye in high throughput screening (HTS) assays due to the fact that its excitation and emission spectra are red-shifted approximately 50 nm relative to fluorescein. Assays based on BODIPY® TMR ligands are therefore less susceptible to interference from tissue auto-fluorescence in the assay matrix, or the effects of colored or fluorescent compounds in the screening libraries. A series of BODIPY® TMR labeled peptides have been prepared that bind to a range of GPCRs including melanin concentrating hormone, bradykinin, and melanocortin receptors. Conditions have been optimized in order to utilize a comparable amount of receptor membrane preparation as is used in a radioligand binding assay. The assays are formatted in 384-well microplates with a standard volume of 40 µL. We have compared the assays across the different fluorescence polarization (FP) readers available to determine the parameters for each instrument necessary to achieve the required precision.     

A reporter assay for G-protein-coupled receptors using a B-cell line suitable for stable episomal expression

We have established a cAMP response element (CRE)-mediated reporter assay system for G-protein-coupled receptors (GPCRs) using an oriP-based estrogen-inducible expression vector and the B-cell line (GBC53 or GBCC71) that expresses EBNA-1 and is adapted to serum-free culture. GBC53 harbors a GAL4-ER expression unit and a CRE-luciferase gene in the genome, and GBCC71 also harbors expression units for two chimeric Gαs proteins (Gs/q and Gs/i). Introduction of a GPCR expression plasmid into GBC53 or GBCC71 creates polyclonal stable transformants in 2 weeks, and these are easily expanded and used for assays after induction of the GPCR expression. Using GBC53, we detected ligand-dependent signals of Gs-coupled GPCRs such as glucagon-like peptide 1 receptor (GLP1R) and β2 adrenergic receptor (β2AR) with high sensitivity. Interestingly, we also detected constitutive activity of β2AR. Using GBCC71, we detected ligand-dependent signals of Gq- or Gi-coupled GPCRs such as H1 histamine receptor and CXCR1 chemokine receptor in addition to Gs-coupled GPCRs. An agonist, antagonist, or inverse agonist was successfully evaluated in this system. We succeeded in constructing a 384-well high-throughput screening (HTS) system for GLP1R. This system enabled us to easily and rapidly make a large number of efficient GPCR assay systems suitable for HTS as well as ligand hunting of orphan GPCRs.     

Miniaturization of a Mammalian Cell-Based Assay: Luciferase Reporter Gene Readout in a 3 Microliter 1536-Well Plate

The combined efforts of the fields of combinatorial chemistry and genomics have significantly increased the number of compounds and therapeutic targets available for screening. The number of compounds will reach into the million range in the near future and provide vast chemical diversity for drug discovery. However, this reservoir of chemical diversity creates downstream hurdles for any screening effort. Properly examining this number of compounds increases investments dramatically, both in the number of dollars spent and amount of limited reagents depleted. Traditional HTS techniques, such as the use of 96-well microtiter plates, have paved the way for faster processing speeds, but are being rapidly overwhelmed by screening demands. Miniaturization of such assays will allow for greater throughput, while concurrently reducing cost. To date, miniaturization efforts have been most successfully applied to bacterial and soluble protein based assays. Questions about the ability to deliver microquantities of mammalian cells without disruption of the cell membrane and/or activation of stress responses have been raised. An assay has been developed in which a human T-cell screen has been adapted to a 1536-well plate format. Through the use of a luciferase reporter gene system, it is shown that a mammalian cell-based assay may be successfully performed in 3 μl and potent inhibitors of the target of interest identified.     

Analysis of protein-peptide interaction by a miniaturized fluorescence polarization assay using cyclin-dependent kinase 2/cyclin E as a model system.

As a result of the increasing size of chemical libraries, more rapid and highly sensitive strategies are needed to accelerate the process of drug discovery without increasing the cost. One means of accomplishing this is to miniaturize the assays that enter high-throughput screening (HTS). Miniaturization requires an assay design that has few steps, has a large degree of separation between the signal and background, and has a low well to well signal variation. Fluorescence polarization (FP) is an assay type that, in many cases, meets all of the above requirements. FP is a homogenous method that allows interactions between molecules to be measured directly in solution. This article demonstrates the application of FP in a miniaturized HTS format, using 1536-well plates, to measure direct binding between cyclin-dependent kinase 2/cyclin E complex (CDK2/E) and an 8-mer-peptide kinase inhibitor. The data indicate that low variability and high specificity allow rapid and precise identification of antagonist compounds affecting CDK2/E-peptide interactions.     

Readout technologies for highly miniaturized kinase assays applicable to high-throughput screening in a 1536-well format

This article discusses the development of homogeneous, miniaturized assays for the identification of novel kinase inhibitors from very large compound collections. In particular, the suitability of time-resolved fluorescence resonance energy transfer (TR-RET) based on phospho-specific antibodies, an antibody-independent fluorescence polarization (FP) approach using metal-coated beads (IMAP technology), and the determination of adenosine triphosphate consumption through chemiluminescence is evaluated. These readouts are compared with regard to assay sensitivity, compound interference, reagent consumption, and performance in a 1536-well format, and practical considerations for their application in primary screening or in the identification of kinase substrates are discussed. All of the tested technologies were found to be suitable for miniaturized high-throughput screening (HTS) in principle, but each of them has distinct limitations and advantages. Therefore, the target-specific selection of the most appropriate readout technology is recommended to ensure maximal relevance of HTS campaigns.     

A simple, no-wash cell adhesion-based high-throughput assay for the discovery of small-molecule regulators of the integrin CD11b/CD18

The leukocyte-specific integrin CD11b/CD18 plays a key role in the biological function of these cells and represents a validated therapeutic target for inflammatory diseases. Currently, the low affinity interaction between CD11b/CD18 integrin and its respective ligand poses a challenge in the development of cell-based adhesion assays for the high-throughput screening (HTS) environment. Here the authors describe a simple cell-based adhesion assay that can be readily used for HTS for the discovery of functional regulators of CD11b/CD18. The assay consistently produces acceptable Z' values (> 0.5) for HTS. After testing the assay using 2 established blocking antibodies as reference biologicals, the authors performed a proof-of-concept primary screen using a library of 6612 compounds and identified both agonist and antagonist hits.     

Ultra-high throughput screen of two-million-member combinatorial compound collection in a miniaturized, 1536-well assay format

Results of a complete survey of the more than 2-million-member Pharmacopeia compound collection in a 1536-well microvolume screening assay format are reported. A complete technology platform, enabling the performance of ultra-high throughput screening in a miniaturized 1536-well assay format, has been assembled and utilized. The platform consists of tools for performing microvolume assays, including assay plates, liquid handlers, optical imagers, and data management software. A fluorogenic screening assay for inhibition of a protease enzyme target was designed and developed using this platform. The assay was used to perform a survey screen of the Pharmacopeia compound collection for active inhibitors of the target enzyme. The results from the survey demonstrate the successful implementation of the ultra-high throughout platform for routine screening purposes. Performance of the assay in the miniaturized format is equivalent to that of a standard 96-well assay, showing the same dependence on kinetic parameters and ability to measure enzyme inhibition. The survey screen identified an active class of compounds within the Pharmacopeia compound collection. These results were confirmed using a standard 96-well assay.     

TR-FRET-based high-throughput screening assay for identification of UBC13 inhibitors

UBC13 is a noncanonical ubiquitin conjugating enzyme (E2) that has been implicated in a variety of cellular signaling processes due to its ability to catalyze formation of lysine 63-linked polyubiquitin chains on various substrates. In particular, UBC13 is required for signaling by a variety of receptors important in immune regulation, making it a candidate target for inflammatory diseases. UBC13 is also critical for double-strand DNA repair and thus a potential radiosensitizer and chemosensitizer target for oncology. The authors developed a high-throughput screening (HTS) assay for UBC13 based on the method of time-resolved fluorescence resonance energy transfer (TR-FRET). The TR-FRET assay combines fluorochrome (Fl)-conjugated ubiquitin (fluorescence acceptor) with terbium (Tb)-conjugated ubiquitin (fluorescence donor), such that the assembly of mixed chains of Fl- and Tb-ubiquitin creates a robust TR-FRET signal. The authors defined conditions for optimized performance of the TR-FRET assay in both 384- and 1536-well formats. Chemical library screens (total 456 865 compounds) were conducted in high-throughput mode using various compound collections, affording superb Z' scores (typically >0.7) and thus validating the performance of the assays. Altogether, the HTS assays described here are suitable for large-scale, automated screening of chemical libraries in search of compounds with inhibitory activity against UBC13.