Ultra-high-throughput screening for antagonists of a Gi-coupled receptor in a 2.2-microl 3,456-well plate format cyclicAMP assay

3',5'-Cyclic adenosine monophosphate (cAMP) is a common intracellular second messenger that enables cells to respond to external stimuli. Measurement of intracellular cAMP concentrations is thus widely used for studying guanosine triphosphate binding protein-coupled receptors (GPCRs), which make up a large class of pharmaceutical drug targets. Although several assay technologies exist to measure cAMP, most are not suitable for ultra-high-throughput screening (uHTS), as is often required for screening large (greater than 1 million) chemical libraries for the identification of suitable leads for drug development. Here we report that the enzyme fragment complementation assay, a homogeneous gain of signal assay based on complementation of two fragments of a beta-galactosidase enzyme, is compatible with uHTS requirements of a 2.2-microl total assay volume in 3,456-well plate format. We describe the miniaturization of this assay into 3,456-well plate format exhibiting comparable sensitivity and plate statistics to those of a 384-well assay and the application of this assay in uHTS for the identification of antagonists of a Gi-coupled receptor.     

Miniaturization and automation of an ubiquitin ligase cascade enzyme-linked immunosorbent assay in 1,536-well format

Enzyme-linked immunosorbent assays (ELISAs) are a long established and widely used assay format for drug discovery and diagnostics. They offer many advantages over homogeneous assay formats, including high sensitivity and separation (wash) steps that remove detection-interfering compounds. Many high-throughput screening assays are now performed in miniaturized formats (1,536- and 3,456-well plates) for higher throughput and lower reagent consumption. With miniaturization, separation steps in assays such as ELISA can become difficult to implement. Here we report on the implementation of the Kalypsys, Inc. (San Diego, CA) 1,536-well plate washer to enable the successful miniaturization and full automation of an ELISA that monitors ubiquitin ligase activity. The 1,536-well plate ELISA was robust and used for the high-throughput screening of a large screening collection (>1 million compounds).     

A multi-modality assay platform for ultra-high throughput screening

The demand for increased throughput during primary screening using less reagents is changing the way of drug discovery. Searching for hits using high throughput screening in 96-well format plates is being replaced by the use of higher density plates, such as 384 and 1536-well formats. The analysis of radiometric assays by scintillation counters is becoming limiting since only 12 wells can be counted at a time. Charged coupled device (CCD) camera based instruments, that image the whole plate in one exposure, speed up detection and are compatible with any microplate footprint. Researchers are also demanding a choice of detection methods, including fluorescence, luminescence and radioactivity, and require imagers suitable for all applications. LEADseeker Homogenous Imaging System is a multi-modality platform offering imaging technology and assay toolboxes for radiometric, fluorescent and luminescent based assays. LEADseeker allows the very rapid analysis of high density formats enabling ultra-high throughput screening of a range of biological assays. Research areas that can be studied using this system include enzyme assays, receptor binding and molecular interactions.     

Real-time and high throughput monitoring of cAMP in live cells using a fluorescent membrane potential-sensitive dye

Adenosine-3',5'-cyclic monophosphate (cAMP) conveys the signals from G-protein coupled receptors (GPCRs) and regulates a variety of downstream cellular events. However, there are few robust assays available for measuring cAMP in live cells. Most of the existing cAMP assays require cell lysis and/or have relatively low throughput. We report a live cell-based cAMP assay that has been developed to record the real-time changes in intracellular cAMP. By employing a mutated cyclic-nucleotide-gated ion channel (CNGC) as the cAMP biosensor, the change of cAMP level is coupled to the change of transmembrane potential that is measured through a new fluorescent membrane potential (MP)-sensitive dye, HLB 021-152. We have successfully used HLB 021-152 for homogeneously monitoring cAMP stimulations in live cells under both serum-containing and serum-free environments. Upon stimulating the endogenous or heterogenous GPCRs on CNGC-cloned human embryonic kidney 293 cells with agonists, the fluorescence signal of HLB 021-152 increases rapidly. It has much greater assay dynamic range than DiSBAC2(3), the existing "gold standard" dye for measuring cellular MP. This new MP dye can be readily formulated for high throughput screening of GPCR modulators either with serum or without serum.     

Characterization of CHO cells stably expressing a G alpha 16/z chimera for high throughput screening of GPCRs

G protein-coupled receptors (GPCRs) are important therapeutic targets for drug discovery. The identification and characterization of new ligands ideally requires the use of high throughput assays that are applicable to all GPCR subtypes. To circumvent the problem of different GPCRs coupling to distinct intracellular second messenger pathways, we describe a new method that uses the chimeric Galpha protein 16z25 to facilitate this process. Stably expressed in Chinese hamster ovary cells, 16z25 allows G(i/o)- and G(s)-coupled receptors to mobilize intracellular Ca(2+) upon agonist stimulation. We have generated nine cell lines each stably expressing 16z25 and a GPCR. All cell lines respond to appropriate agonist stimulation in fluorometric imaging plate reader (FLIPR) assays with robust and potent Ca(2+) mobilization. Several of these lines have been pharmacologically characterized using agonists and antagonists. We also demonstrate that the coexpression of GPCR and 16z25 does not interfere with the receptors' ability to activate endogenous signaling pathways. The ability of 16z25 to functionally mediate the agonist stimulation of a broad spectrum of GPCRs indicates that the use of cell lines stably coexpressing this chimera and GPCRs will simplify the drug screening process and aid in the deorphanization of new receptors.     

啤酒酵母等表达的G蛋白偶联受体在药物高通量筛选中的应用

G蛋白偶联受体具有重要的生理功能和病理意义,已成为一类重要的药物靶点。针对靶标G蛋白偶联受体从大量化合物中筛选出理想的药物,就需要开发特异且灵敏的高通量筛选体系。真核酵母细胞具有易操作、培养条件简单稳定、基因组学研究透彻、外源蛋白表达工具丰富多样、检测分析方法成熟等优点,使它成为一种理想的药物筛选工具。文章对酵母表达G蛋白偶联受体用于构建药物高通量筛选模型的研究现状和发展前景做综述。     

A bioluminescent-based, HTS-compatible assay to monitor G-protein-coupled receptor modulation of cellular cyclic AMP

We have developed a novel assay for monitoring changes in intracellular cyclic AMP (cAMP) concentration with high sensitivity (30 +/- 5 fmol [mean +/- standard error of the mean] of cAMP per well) and reproducibility (Z' of > 0.8). The assay is of format amenable to high throughput screening (HTS) in 96-, 384-, and 1,536-well plates, and as a bioluminescent assay is potentially less prone to interferences originating from fluorescent compounds. Because of its high sensitivity, fewer numbers of cells (1,000 cells per well) in low-volume 384-well plates are required to screen for changes in cAMP concentrations. The assay does not rely on the use of antibodies, and thus it does not suffer from changes in the affinity or quality of the antibodies. The assay is based on the fact that cAMP is a potent activator of cAMP-dependent protein kinase (PKA), and activation of PKA can be monitored by measuring ATP utilization in a kinase reaction. The amount of ATP consumed can be measured using a luciferase/luciferin luminescent reaction. Since the amount of relative luminescence units (RLU) generated is a measure of the remaining ATP, a reciprocal relationship between RLU and both the activity of PKA and the intracellular concentration of cAMP is observed. Thus, the functional activity of agents that modulate the activity of Galpha(s) or Galpha(i) forms of G-protein-coupled receptors (GPCRs), which cause change in intracellular cAMP, can be monitored by the change in the activity of PKA and the amount of RLU readout. The assay can be performed in two steps and requires only 30 min after cell lysis for completion. The assay has been successfully used to generate 50% effective concentration (EC(50)) values for forskolin, a known direct activator of cellular adenylate cyclases, and EC(50) values for agonists and 50% inhibitory concentration values for antagonists modulating GPCRs that alter adenylate cyclase activity (Galpha(s) and Galpha(i)). Finally, adherent, suspension, and frozen cells have been successfully used in this assay, thus offering flexibility and convenience for many HTS applications.     

Validation of an atomic absorption rubidium ion efflux assay for KCNQ/M-channels using the ion Channel Reader 8000

M-channels (M-current), encoded by KCNQ2/3 K(+) channel genes, have emerged as novel drug targets for a number of neurological disorders. The lack of direct high throughput assays combined with the low throughput of conventional electrophysiology (EP) has impeded rapid screening and evaluation of K(+)-channel modulators. Development of a sensitive and efficient assay for the direct measurement of M-current activity is critical for identifying novel M-channel modulators and subsequent investigation of their therapeutic potential. Using a stable CHO cell line expressing rat KCNQ2/3 K(+) channels confirmed by EP, we have developed and validated a nonradioactive rubidium (Rb(+)) efflux assay in a 96-well plate format. The Rb(+) efflux assay directly measures the activity of functional channels by atomic absorption spectroscopy using the automated Ion Channel Reader (ICR) 8000. The stimulated Rb(+) efflux from KCNQ2/3-expressing cells was blocked by the channel blockers XE991 and linopirdine with IC(50) values of 0.15 microM and 1.3 microM, respectively. Twelve compounds identified as KCNQ2/3 openers were further assessed in this assay, and their EC(50) values were compared with those obtained with EP. A higher positive correlation coefficient between these two assays (r = 0.60) was observed than that between FlexStation membrane potential and EP assays (r = 0.23). To simplify the assay and increase the throughput, we demonstrate that EC(50) values obtained by measuring Rb(+) levels in the supernatant are as robust and consistent as those obtained from the ratio of Rb(+) in supernatant/lysate. By measuring the supernatant only, the throughput of ICR8000 in an eight-point titration is estimated to be 40 compounds per day, which is suitable for a secondary confirmation assay.     

Configuration of a scintillation proximity assay for the activity assessment of recombinant human adenine phosphoribosyltransferase

Adenine phosphoribosyltransferase plays a role in purine salvage by catalyzing the direct conversion of adenine to adenosine monophosphate. The involvement of the purine salvage pathway in tumor proliferation and angiogenesis makes adenine phosphoribosyltransferase a potential target for oncology drug discovery. We have expressed and characterized recombinant, N-terminally His-tagged human adenine phosphoribosyltransferase. Two assay formats were assessed for use in a high throughput screen: a spectrophotometric-based enzyme-coupled assay system and a radiometric ionic capture scintillation proximity bead assay format. Ultimately, the scintillation proximity assay format was chosen because of automated screening compatibility limitations of the coupled assay. We describe here the biochemical characterization of adenine phosphoribosyltransferase and the development of a robust, homogeneous, 384-well assay suitable for high throughput screening.     

Development of a cyclic adenosine monophosphate assay for Gi-coupled G protein-coupled receptors by utilizing the endogenous calcitonin activity in Chinese hamster ovary cells

Activation of G(i)-coupled G protein-coupled receptor (GPCRs) by their ligands leads to inhibition of adenylyl cyclase (AC) and reduction of cyclic adenosine monophosphate (cAMP) levels in cells. The traditional cAMP assay for G(i)-coupled GPCRs commonly uses forskolin, a nonspecific AC activator, to increase the basal cAMP level in cells to create an assay window for ligand detection. However, there is still a need to develop a nonforskolin-based cAMP assay because of the challenges inherent in titrating the concentration of forskolin to achieve a reliable assay window, along with issues related to the cAMP-independent effects of forskolin. Herein, we describe such an assay by utilizing the endogenous activity of the calcitonin receptor in Chinese hamster ovary (CHO) cells. The calcitonin receptor is a G(s)-coupled GPCR that, when activated by calcitonin, leads to the stimulation of AC and increases cAMP in cells. Thus, we use calcitonin, instead of forskolin, to increase the basal cAMP level in CHO cells to achieve an assay window. We demonstrated that calcitonin peptides robustly increased cAMP accumulation in several CHO cell lines stably expressing well-known G(i)-coupled GPCRs, such as the Dopamine D2 receptor, the Opioid μ receptor, or the Cannabinoid receptor-1. Agonists of these G(i)-coupled GPCRs attenuated calcitonin-induced cAMP production in their receptor stable cell lines. On the other hand, antagonists and/or inverse agonists blocked the effects of their agonists on calcitonin-induced cAMP production. This calcitonin-based cAMP assay has been demonstrated to be sensitive and robust and exhibited acceptable assay windows (signal/noise ratio) and, thus, can be applied to screen for agonists and antagonists/inverse agonists of G(i)-coupled GPCRs in high-throughput screening formats.     

Techniques: how to boost GPCR mutagenesis studies using yeast

G-protein-coupled receptors (GPCRs) are the major targets of today's medicines. To elucidate the mechanism of activation of GPCRs and the interaction of these receptors with their G proteins, mutagenesis studies have proven to be a powerful tool and have provided insight into the structure and function of GPCRs. Random mutagenesis is useful in this respect particularly when combined with a robust screening assay that is based on the functional properties of the mutants. In this article, the use of random mutagenesis combined with a functional screening assay in yeast is described and compared with alternative approaches such as site-directed mutagenesis per se, alanine/cysteine scanning and another screening assay, receptor selection and amplification technology (R-SAT). Screening in yeast of randomly mutated GPCRs has proven successful in the identification of ligands for orphan receptors and in high-throughput approaches. Moreover, it has provided substantial insight into G-protein coupling and receptor activation.     

A fluorescence-based thiol quantification assay for ultra-high-throughput screening for inhibitors of coenzyme A production

Here we report the development and miniaturization of a cell-free enzyme assay for ultra-high-throughput screening (uHTS) for inhibitors of two potential drug targets for obesity and cancer: fatty acid synthase (FAS) and acetyl-coenzyme A (CoA) carboxylase (ACC) 2. This assay detects CoA, a product of the FAS-catalyzed condensation of malonyl-CoA and acetyl-CoA. The free thiol of CoA can react with 7-diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin (CPM), a profluorescent coumarin maleimide derivative that becomes fluorescent upon reaction with thiols. FAS produces long-chain fatty acid and CoA from the condensation of malonyl-CoA and acetyl-CoA. In our FAS assay, CoA released in the FAS reaction forms a fluorescence adduct with CPM that emits at 530 nm when excited at 405 nm. Using this detection method for CoA, we measured the activity of sequential enzymes in the fatty acid synthesis pathway to develop an ACC2/FAS-coupled assay where ACC2 produces malonyl-CoA from acetyl-CoA. We miniaturized the FAS and ACC2/FAS assays to 3,456- and 1,536-well plate format, respectively, and completed uHTSs for small molecule inhibitors of this enzyme system. This report shows the results of assay development, miniaturization, and inhibitor screening for these potential drug targets.     

A 1,536-well [(35)S]GTPgammaS scintillation proximity binding assay for ultra-high-throughput screening of an orphan galphai-coupled GPCR

Members of the superfamily of seven transmembrane receptors, known as G protein-coupled receptors (GPCRs), are important targets for many therapeutic areas in drug discovery. A homogeneous guanosine 5'-O-(3-[(35)S]thio)triphosphate ([(35)S]GTPgammaS) scintillation proximity assay (SPA) binding assay targeting a Galphai-coupled GPCR recombinantly expressed in membranes of Chinese hamster ovary (CHO) cells was developed and miniaturized into 1,536-well plate format. The primary ultra-high-throughput screen of the entire compound collection was accomplished on the Kalypsys (San Diego, CA) robotic platform at a concentration of 8 muM using the 1,536-well [(35)S]GTPgammaS SPA binding functional assay. The signal-to-noise ratio of the primary screen was approximately 2.1-fold, and the plate coefficient of variation for the compound field was approximately 11%. The hit rate from the primary screen for receptor agonists at >35% activity was approximately 0.3%. Primary hits were cherry-picked, confirmed in triplicate, counterscreened against untransfected CHO cell membranes, and further analyzed in a cyclic AMP functional assay, resulting in 34 leads for optimization.     

A bioluminescent cytotoxicity assay for assessment of membrane integrity using a proteolytic biomarker

Measurement of cell membrane integrity has been widely used to assess chemical cytotoxity. Several assays are available for determining cell membrane integrity including differential labeling techniques using neutral red and trypan blue dyes or fluorescent compounds such as propidium iodide. Other common methods for assessing cytotoxicity are enzymatic “release” assays which measure the extra-cellular activities of lactate dehydrogenase (LDH), adenylate kinase (AK), or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in culture medium. However, all these assays suffer from several practical limitations, including multiple reagent additions, scalability, low sensitivity, poor linearity, or requisite washes and medium exchanges. We have developed a new cytotoxicity assay which measures the activity of released intracellular proteases as a result of cell membrane impairment. It allows for a homogenous, one-step addition assay with a luminescent readout. We have optimized and miniaturized this assay into a 1536-well format, and validated it by screening a library of known compounds from the National Toxicology Program (NTP) using HEK 293 and human renal mesangial cells by quantitative high-throughput screening (qHTS). Several known and novel membrane disrupters were identified from the library, which indicates that the assay is robust and suitable for large scale library screening. This cytotoxicity assay, combined with the qHTS platform, allowed us to quickly and efficiently evaluate compound toxicities related to cell membrane integrity.     

A novel assay of Gi/o-linked G protein-coupled receptor coupling to potassium channels provides new insights into the pharmacology of the group III metabotropic glutamate receptors

The group III metabotropic glutamate receptors (mGluRs) represent a family of presynaptically expressed G-protein-coupled receptors (GPCRs) with enormous therapeutic potential; however, robust cellular assays to study their function have been difficult to develop. We present here a new assay, compatible with traditional high-throughput screening platforms, to detect activity of pharmacological ligands interacting with G(i/o)-coupled GPCRs, including the group III mGluRs 4, 7, and 8. The assay takes advantage of the ability of the Gbetagamma subunits of G(i) and G(o) heterotrimers to interact with G-protein regulated inwardly rectifying potassium channels (GIRKs), and we show here that we are able to detect the activity of multiple types of pharmacophores including agonists, antagonists, and allosteric modulators of several distinct GPCRs. Using GIRK-mediated thallium flux, we perform a side-by-side comparison of the activity of a number of commercially available compounds, some of which have not been extensively evaluated because of the previous lack of robust assays at each of the three major group III mGluRs. It is noteworthy that several compounds previously considered to be general group III mGluR antagonists have very weak activity using this assay, suggesting the possibility that these compounds may not effectively inhibit these receptors in native systems. We anticipate that the GIRK-mediated thallium flux strategy will provide a novel tool to advance the study of G(i/o)-coupled GPCR biology and promote ligand discovery and characterization.     

Confocal fluorescence detection expanded to UV excitation: the first continuous fluorimetric assay of human steroid sulfatase in nanoliter volume

Steroid sulfatase is an enzyme that currently enjoys considerable interest as a potential drug target in the treatment of estrogen- and androgen-dependent diseases, in particular breast cancer. We have purified human steroid sulfatase to apparent homogeneity from recombinant Chinese hamster ovary cells, and we established an assay with a new fluorogenic substrate, 3,4-benzocoumarin-7-O-sulfate (1). Substrate 1 features a K(m) value of 22.5 microM, which is close to the value for the natural substrate dehydroepiandrosterone sulfate (26 microM) and much lower than the K(m) values of other synthetic substrates (276-736 microM). Importantly, the cleavage of substrate 1 can be monitored continuously during the enzymatic cleavage, since a change in fluorescence intensity is detectable at the pH where the enzyme is active; in contrast, all other synthetic substrates described so far require alkalization to reveal a measurable absorbance or fluorescence signal. The adaptation of the assay to the 96-well format allows continuous monitoring of multiple wells in a microplate fluorescence reader. Applications of the assay for the determination of IC(50) and K(i) values of novel steroid sulfatase inhibitors are presented. Most importantly the assay was transferred to the nanoscale format (1-microl assay volume) in 2080-well plates with confocal fluorescence detection. This miniaturization will permit screening with a minimum throughput of 20000 compounds per day. The system presented demonstrates that the confocal detection platform used for nanoscreening can be successfully adapted to assays for which conventional ultraviolet dyes like coumarins are necessary. This strongly broadens the application range of confocal readers in drug screening.     

Well-less,gel-permeation formats for ultra-HTS

An alternative approach to miniaturization has been developed that does not rely upon increasing microtiter plate densities. Gel-permeation assays, originally developed for screening antibody–antigen interactions and anti-microbial screening, are a well-less technique. Newer approaches extending this technology to enable the screening of traditional compound-collections show exceptional promise and offer the potential to convert most (if not all) 96-well assays into this novel high-density format.     

Allosteric modulation of G protein-coupled receptors

G protein-coupled receptors (GPCRs) constitute the largest receptor superfamily in the human genome and represent the most common targets of drug action. Classic agonist and antagonist ligands that act at GPCRs tend to bind to the receptor's orthosteric site, that is, the site recognized by the endogenous agonist for that receptor. However, it is now evident that GPCRs possess additional, extracellular, allosteric binding sites that can be recognized by a variety of small molecule modulator ligands. Allosteric modulators offer many advantages over classic orthosteric ligands as therapeutic agents, including the potential for greater GPCR-subtype selectivity and safety. However, the manifestations of allosterism at GPCRs are many and varied and, in the past, traditional screening methods have generally failed to detect many allosteric modulators. More recently, there have been a number of major advances in high throughput screening, including the advent of cell-based functional assays, which have led to the discovery of more allosteric modulator ligands than previously appreciated. In addition, a number of powerful analytical techniques have also been developed exclusively for detecting and quantifying allosteric effects, based on an increased awareness of various mechanisms underlying allosteric modulator actions at GPCRs. Together, these advances promise to change the current paucity of GPCR allosteric modulators in the clinical setting and yield novel therapeutic entities for the treatment of numerous disorders.