Showing papers in "Assay and Drug Development Technologies in 2006"

Label-free cell-based assays with optical biosensors in drug discovery.

Abstract: Once viewed solely as a tool for low throughput and kinetic analysis of biomolecular interactions, optical biosensors are gaining widespread uses in drug discovery because of recent advances in instrumentation and experimental design. These advances have expanded the capabilities of optical biosensors to meet the needs at many points in the drug discovery process. Concurrent shifts in drug discovery paradigms have seen the growing use of whole cell systems for drug screens, thus creating both a need in drug discovery and a solution in optical biosensors. This article reviews important advances in optical biosensor instrumentation, and highlights the potential of optical biosensors for drug discovery with an emphasis on whole cell sensing in both high throughput and high content fashions.

Dynamic and label-free cell-based assays using the real-time cell electronic sensing system.

Abstract: Cell-based assays have become an integral part of the preclinical drug development process. Recently, noninvasive label-free cell-based assay technologies have taken center stage, offering important and distinct advantages over and in addition to traditional label-based endpoint assays. Dynamic monitoring of live cells, the preclusion of label, and kinetics are some of the fundamental features of cell-based label-free technologies. In this article we will discuss the real-time cell electronic sensing (RT-CES, ACEA Biosciences Inc., San Diego, CA) system and some of its key applications for cell-based assays such as cell proliferation and cytotoxicity, functional assays for receptor-ligand analysis, cell adhesion and spreading assays, dynamic monitoring of endothelial barrier function, and dynamic monitoring of cell migration and invasion. Also, where appropriate we will briefly discuss other label-free technologies in an application-specific manner.

Cellular dielectric spectroscopy: a label-free comprehensive platform for functional evaluation of endogenous receptors.

Abstract: The CellKey™* (MDS Sciex, South San Francisco, CA) system enables comprehensive pharmacological evaluation of cell surface receptors, including G-protein coupled receptors (GPCRs) and tyrosine kinase receptors, using adherent and suspension cell lines and primary cells. A unique application enabled by the ability of the CellKey system to reliably quantify activation of endogenous receptors is receptor panning. This application allows investigators to easily screen disease-relevant cell types for functionally active target receptors by treating cells with a panel of receptor-specific ligands. Receptor panning of multiple cell types including Chinese hamster ovary, human embryonic kidney 293, HeLa, U-937, U-2 OS, and TE671 cells resulted in the identification of many functionally active, differently coupled endogenous GPCRs, some of which have not been previously documented in the literature. Upon detecting GPCR activation in live cells, unique cellular dielectric spectroscopy (CDS) response profiles are ge...

Live Cell Quality Control and Utility of Real-Time Cell Electronic Sensing for Assay Development

Abstract: In this paper we have explored the utility of the real-time cell electronic sensing (RTCES™, ACEA Biosciences Inc., San Diego, CA) system for monitoring the quality of live cells in cell-based assays as well as for assay development. We have demonstrated that each cell type displays unique growth kinetic profiles that provide a quantitative account of cell behavior and can be used as a diagnostic tool for cellular quality control. The utility of the specific signature patterns was shown by demonstrating the significant differences in primary cell behavior depending on the supplier. In addition, the RT-CES system was able to differentiate cell behavior depending on the passage stage of the cells. The utility of the RT-CES system as an assay development tool was demonstrated in cytotoxicity assays. The RT-CES system not only provides information regarding the potency of cytotoxic compounds, but in addition relates potency to the rate of the response for each concentration of the compound tested, which is im...

Application of Laser-Scanning Fluorescence Microplate Cytometry in High Content Screening

Abstract: The resolution of cell-based assays down to the cellular level has created new opportunities for the drug discovery process. Aptly named high content analysis, such an approach is enabling new methods of analysis for the broad range of therapeutic targets emerging in the post-genomics era, and offering alternative multiparametric readouts for some traditional analyses. Microplate cytometry is one of the technologies that is being applied to a broad range of assays utilizing fluorescent labeling, at throughputs compatible with primary screening campaigns. Cellular resolution is achieved using scanning laser excitation coupled to photomultiplier detection. This configuration results in area-based scanning across a large field of view, plus simultaneous detection of up to four emission colors for efficient multiplexing. Microplate cytometry is being used most extensively in the field of oncology research because of its usefulness for numerous applications, including protein kinase activity, cell cycle analysis, and cell colony formation. The review focuses on the Acumen Explorer microplate cytometer (TTP LabTech Ltd., Melbourn, Hertfordshire, UK), detailing the principal components of the instrument and providing an overview of its use in high content screening.

Dynamic detection of natural killer cell-mediated cytotoxicity and cell adhesion by electrical impedance measurements.

Abstract: Measurement of electrical impedance is a relatively new real-time and label-free method for monitoring cell adhesive properties. Impedance measurements are performed in tissue culture wells in which the bottom is equipped with gold electrodes. The extent of electrode coverage by living cells as well as the strength of the bond between the cell membrane and the electrode surface determines the impedance, which in real-time cell electrical sensing (RT-CES, ACEA Biosciences, San Diego, CA) is measured as the cell index (CI). We showed for carcinoma cells that CI was linearly correlated to the number of cells and that CI also was related to the amount of coating (laminin-5) of the wells. When natural killer (NK) cells were added to adherent carcinoma cells (target cells) CI declined rapidly dependent on the NK cell:target cell ratio. The initial decrease of CI was much more pronounced than target cell death as measured by [(3)H]thymidine incorporation assay. Such a rapid fall of CI was due to changes in the adhesion and morphology of target cell undergoing apoptosis. It took more than 6 h before the extent of cell death and fall of CI were comparable. We also showed using A431 cells and an antibody specific for the human epidermal growth factor receptor (Erbitux, manufactured by Merck KGaA, Darmstadt, Germany) that RT-CES could be used to monitor antibody-dependent cellular cytotoxicity. Thus RT-CES is a convenient way to continuously determine cell number and cell adhesion and may offer early detection of NK cell-mediated cytotoxic effects.

Transporter assays using solid supported membranes: a novel screening platform for drug discovery.

Abstract: Transporters are important targets in drug discovery. However, high throughput-capable assays for this class of membrane proteins are still missing. Here we present a novel drug discovery platform technology based on solid supported membranes. The functional principles of the technology are described, and a sample selection of transporter assays is discussed: the H+-dependent peptide transporter PepT1, the gastric proton pump, and the Na+/Ca2+ exchanger. This technology promises to have an important impact on the drug discovery process.

Evaluation of MTT Assay for Measurement of Emodin-Induced Cytotoxicity

Abstract: The specificity and sensitivity of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) tetrazolium assay can be influenced by certain factors, such as colored substances and cell volume. When the MTT assay is applied to measure cytotoxicity induced by emodin, its accuracy might be affected by emodin itself. Vascular smooth muscle cells were cultured in M199 medium. The optical density of emodin or formazan was measured by spectrophotometry. Emodin has a different absorption spectrum in different solvents. The solvents containing water induced a red shift of the absorption curve of emodin, which increased the overlap of the absorption curves of emodin and formazan. Formazan was formed from the MTT tetrazolium salt by emodin in a dosedependent manner, which was partially suppressed by serum. Cytotoxicity was induced by emodin in a time- and dose-dependent manner in a modified MTT assay. The data suggest that emodin can alter the accuracy of the MTT assay but that a modified MTT assay is s...

A high-capacity membrane potential FRET-based assay for NaV1.8 channels.

Abstract: Clinical treatment of neuropathic pain can be achieved with a number of different drugs, some of which interact with all members of the voltage-gated sodium channel (NaV1) family. However, block of central nervous system and cardiac NaV1 channels can cause dose-limiting side effects, preventing many patients from achieving adequate pain relief. Expression of the tetrodotoxinresistant NaV1.8 subtype is restricted to small-diameter sensory neurons, and several lines of evidence indicate a role for NaV1.8 in pain processing. Given these features, NaV1.8 subtypeselective blockers are predicted to be efficacious in the treatment of neuropathic pain and to be associated with fewer adverse effects than currently available therapies. To facilitate the identification of NaV1.8-specific inhibitors, we stably expressed the human NaV1.8 channel together with the auxiliary human β1 subunit (NaV β1) in human embryonic kidney 293 cells. Heterologously expressed human NaV1.8/NaV β1 channels display biophysical properties...

Evaluation of the rubidium efflux assay for preclinical identification of HERG blockade.

Abstract: Inhibition of the delayed-rectifier potassium channel current, human ether-a-go-go (hERG), by pharmaceutical agents can lead to acquired long QT syndrome and the generation of potentially lethal arrhythmias and sudden death. There remains an unmet need for higher-throughput assays to screen compounds in preclinical development for the potential to block hERG and cause QT prolongation. We evaluated the rubidium efflux assay for its ability to determine block of the hERG potassium channel. hERG-transfected human embryonic kidney-293 cells were cultured on 96-well assay plates and loaded with rubidium ion by incubating in media in which potassium was replaced by 5.4 mM Rb+. Cells were exposed to test compounds and then depolarized with a K+ channel opening buffer containing 50 mM K+. The supernatant was removed, and cells were lysed using 0.1% Triton X-100. Concentration-response curves were generated for test agents by determining the Rb+ efflux using a flame atomic absorption spectrometer. Multiple trials with cisapride yielded 50% inhibitory concentration values between 308.1 +/- 11 nM to 456.3 +/- 24 nM for inhibition of Rb+ efflux and a Z factor of 0.80 +/- 0.07 (n = 5 plates, 12 wells per plate). The values for inhibition of the hERG channel exhibited a rightward shift in potency as compared to those measured using electrophysiological techniques. In addition, we evaluated 19 blinded compounds at 10 microM in the Rb+ efflux assay, and compared results to those using patch clamp electrophysiology and the dofetilide displacement binding assay. The dofetilide displacement binding assay yielded a good correlation with electrophysiological measurements of hERG block. The rubidium efflux assay lacked sensitivity to consistently identify significant channel blockade. In conclusion, the rubidium efflux assay provides a higher-throughput means to identify potent hERG channel blocking agents, but lacks the sensitivity required to accurately determine the potency of blockade.

Interrogating genomes with combinatorial artificial transcription factor libraries: asking zinc finger questions.

Abstract: Artificial transcription factors (ATFs) are proteins designed to specifically bind and regulate genes. Because of their DNA-binding selectivity and modular organization, arrays of zinc finger (ZF) domains have traditionally been used to build the ATF's DNA-binding domains. ATFs have been designed and constructed to regulate a variety of therapeutic targets. Recently, novel combinatorial technologies have been developed to induce expression of any gene of interest or to modify cellular phenotypes. Large repertoires of ATFs have been generated by recombination of all available sequence-specific ZF lexicons. These libraries comprise millions of ATFs with unique DNA-binding specificities. The ATFs are produced by combinatorial assembly of three- and six-ZF building blocks and are linked to activator or repressor domains. Upon delivery into a cell population, any gene in the human genome can potentially be regulated. ATF library members generate genome-wide, experimental perturbations of gene expression, resul...

The use of high-content screening for the discovery and characterization of compounds that modulate mitotic index and cell cycle progression by differing mechanisms of action.

Abstract: The advent of high-content screening has expanded the ability of researchers to identify and quantify compound effects on a number of cellular events in a manner that allows for the rapid screening...

Enzyme fragment complementation binding assay for p38alpha mitogen-activated protein kinase to study the binding kinetics of enzyme inhibitors.

Abstract: The majority of protein kinase assays used in drug discovery research are enzyme activity assays. These assays are based on the measurement of phosphorylated protein or peptide substrate, which is the end product of the enzyme reaction. Since most kinase inhibitors are ATP competitive, prediction of the activity of compounds in cellular systems based on potency values in enzyme activity assays is complex, as this should take into account the affinity of the enzyme for ATP and the cellular ATP concentration. The fact that some of the most successful kinase inhibitors, such as STI 571 (imatinib mesylate, Gleevec, Novartis Pharmaceuticals, East Hanover, NJ), act through binding to the inactive isoform of the kinase provides another limitation of enzyme activity assays. Binding assays allow separate measurement of compound affinity to active and inactive kinase and do not require ATP or substrate in the reaction. Recently, a non-radioactive kinase binding assay for p38 mitogen-activated protein kinase has become available from DiscoveRx (Fremont, CA). The assay method, called HitHunter, utilizes enzyme fragment complementation of Escherichia coli beta-galactosidase to generate an assay signal by chemiluminescence. We have reconfigured the commercial assay kit to study the binding kinetics of two known reference inhibitors of the alpha-isoform of p38, the pyridinyl imidazole SB 203580 and the diaryl urea BIRB 796. Our data confirm the slow association kinetics of BIRB 796 as compared to SB 203580, which corresponded with the requirement of a relatively long preincubation time to obtain maximal effect in a cellular assay. Although neither of the two compounds showed preference for either active or inactive p38alpha, our data demonstrate that the HitHunter kinase binding assay can be used to select compounds that specifically target inactive kinase.

A cellular assay for measuring the modulation of glucose production in H4IIE cells.

Abstract: Type II diabetes and its associated complications are a major health concern of the developed world. One of the hallmarks of diabetes is insulin resistance, where secreted insulin no longer has any effect on its target tissues, namely, liver, muscle, and fat. An important therapeutic strategy is to modulate blood glucose levels using pharmacological agents. Glycogen synthase kinase-3 (GSK3) is a serine-threonine protein kinase that plays important roles in regulating glucose metabolism. It is a key negative regulator of insulin action and is an important contributing factor to insulin resistance in liver, muscle, and adipose tissue. We describe the development of a cell-based assay designed to measure glucose production in rat hepatoma cell line H4IIE liver cells in response to treatment with small molecule inhibitors, including GSK3 inhibitors. The assay is set up in a 96-well format, and glucose production is assessed using a convenient fluorescence-based readout. This disease-relevant cellular assay is...

A Morphology- and Kinetics-Based Cascade for Human Neural Cell High Content Screening

Abstract: The prospect of manipulating endogenous neural stem cells to replace damaged tissue and correct functional deficits represents a novel mechanism for treating a variety of central nervous system disorders. Using human neural precursor cultures and a variety of assays for studying stem cell behavior we have screened two libraries of commercially available compounds using an endpoint high content screening assay. We then performed detailed follow-up mechanistic studies on confirmed hits using endpoint and kinetics assays to characterize and differentiate the mechanisms of action of these compounds. The screening cascade employed successfully identified a number of active compounds with differing mechanisms of action. This approach shows how hits from a phenotypic screen can be prioritized and characterized by high content screening to identify potentially novel mechanisms and druggable targets to take forward into more conventional highthroughput screeening approaches.

A nonadherent cell-based HTS assay for N-type calcium channel using calcium 3 dye.

Abstract: The N-type calcium channel is a member of the voltage-sensitive calcium channel family and plays a major role in the regulation of neurotransmitter release in the central and peripheral nervous systems. Inhibition of the N-type calcium channel by intrathecal administration of the channel-specific blocker omega-conotoxin MVIIA (ziconotide) is efficacious in the treatment of severe chronic pain. While no orally active small molecules that block the N-type calcium channel are currently available, the discovery of such potentially valuable therapeutics would benefit from a reliable, high throughput assay. However, the assay of N-type calcium channel activity by measuring calcium influx using nonadherent cells in a high throughput fashion has not been achieved before, likely owing to a number of technical hurdles. For example, the measurement of calcium levels in nonadherent cells using conventional calcium indicators, such as Fluo-3 or Fluo-4, requires dyeloading the cells in suspension and subsequent removal of extracellular dye. This limits plate throughput and requires constant handling of the cells. To assay the N-type calcium channel activity using a nonadherent cell line in a high throughput manner, we investigated the application of no-wash calcium assay kits from Molecular Devices Corp. (Sunnyvale, CA): FLIPR Calcium, FLIPR Calcium Plus, and FLIPR Calcium 3. We show here that the FLIPR Calcium 3 assay kit can be used with nonadherent IMR-32 cells to measure potassium-evoked, omega-conotoxin MVIIA-reversible calcium flux with high throughput (15,000 data points/day), high quality (Z approximately 0.6), and minimal handling of the cells. Thus, this assay can be used to reliably and efficiently screen large compound libraries in the search for small molecule N-type calcium channel blockers.

A simple cell-based HTS assay system to screen for inhibitors of p53-Hdm2 protein-protein interactions.

Abstract: Green fluorescent protein-assisted readout for interacting proteins (GRIP) is a universal protein interaction discovery system that can be used to generate truly high throughput screening-compatible cellular assays to be used to screen for inhibitors of protein-protein interactions. The technology uses a "bait and prey" principle based on the distinct translocation behavior of the human cyclic AMP phosphodiesterase 4A4. Here we use the p53-Hdm2 Redistribution assay (Fisher BioImage ApS, Soborg, Denmark) as an example to describe the GRIP technology. The p53-Hdm2 Redistribution assay is a high content imaging assay based on the GRIP technology that is designed to measure the interaction between Hdm2 and the tumor suppressor p53. Hdm2 regulates p53 and inhibits its function by modulating its transcriptional activity and stability. Activation of p53 in tumor cells through inhibition of its physical interaction with Hdm2 is therefore a focus of cancer drug discovery. We have performed a pilot screen by screening 3,165 compounds from a diverse small-molecule library for inhibitors of the p53-Hdm2 interaction by using the p53-Hdm2 Redistribution assay. Here we show that by taking advantage of the translocation behavior of nonbound p53, it is possible to identify true inhibitors of the p53-Hdm2 interaction by extracting high content information from the acquired images.

Development of luciferase reporter-based cell assays.

Abstract: Using luciferase reporter constructs driven by specific promoter response elements, we developed a series of stable reporter cell lines for monitoring the activity of specific transcription factors (TFs). These TFs, which play essential roles in regulating diverse biological functions, include nuclear factor κB (NFκB), cyclic AMP response element-binding protein, activator protein 1, signal transducer and activator of transcription 1 and 3, nuclear factor of activated T cells, serum response factor, and hypoxia-inducible factor. The response of the stable reporter cells was highly specific. For example, tumor necrosis factor-α (TNFα) strongly activated NFκB reporter cells, but not other cell lines. The NFκB reporter was active in multiple cell lines, including 293T, HeLa, A549, and NIH3T3 cells, in response to TNFα, indicating that this system is useful to monitor specific TFs in different model cell lines. To facilitate high throughput screening of these cell lines, they were adapted to a 96-well format....

A Homogeneous Cell-Based Assay to Measure Nuclear Translocation Using β-Galactosidase Enzyme Fragment Complementation

Abstract: Positional complementation describes the use of homogeneous assays using β- galactosidase (β gal) enzyme fragment complementation to detect cellular protein translocation. This phenomenon occurs when the protein of interest, recombinantly expressed as a fusion protein with a modified α fragment of β gal, translocates to a cellular compartment expressing an enzyme acceptor fragment of the enzyme. When these fragments interact, high-affinity complementation occurs, and a signal is generated that is then detected upon cell lysis. In the present paper the use of positional complementation is exemplified by measuring nuclear translocation of the glucocorticoid receptor in Chinese hamster ovary-K1 cells. The approach thus provides for homogeneous protocols, in an endpoint microtiter plate assay format, without the use of either imaging or reporter gene techniques. Consequently, these characteristics suggest that the technique is suitable for automated instrumentation protocols used in high throughput screening ...

High-content screening analysis of the p38 pathway: profiling of structurally related p38alpha kinase inhibitors using cell-based assays.

Abstract: The complexity of the p38 mitogen-activated protein kinase (MAPK) signaling pathway presents challenges to understanding the efficacy of p38 inhibitors. Biochemical recombinant kinase assays and tumor necrosis factor α (TNFα) secretion assays are typically used to evaluate p38α inhibitors, but they do not provide insight into proximal intracellular events. Stimulation of the pathway evokes a cascade of phosphorylation events, accompanied by movement of molecules to different cellular compartments. Herein, we describe the profiling and potency comparison of a large set of p38α inhibitors with a pyrimidinone, imidazopyrimidine, or triazolopyrimidine core against biochemical recombinant p38α kinase activity, lipopolysaccharide (LPS)-mediated TNFα secretion by THP-1 cells, and a set of cellular imaging assays in SW1353 chondrocytes and baby hamster kidney cells. These pathway assays included p38 phosphorylation, MAPK-activated protein kinase 2 translocation, and heat shock protein (HSP) 27 phosphorylation. We...

Quantitative characterization of mitosis-blocked tetraploid cells using high content analysis.

Abstract: A range of cellular evidence supporting a G1 tetraploidy checkpoint was obtained from different assay methods including flow cytometry, immunoblotting, and microscopy. Cancer research would benefit if these cellular properties could instead be measured by a single, quantitative, automated assay method, such as high content analysis (HCA). Thus, nocodazole-treated cells were fluorescently labeled for different cell cycle-associated properties, including DNA content, retinoblastoma (Rb) and histone H3 phosphorylation, p53 and p21WAF1 expression, nuclear and cell sizes, and cell morphology, and automatically imaged, analyzed, and correlated using HCA. HCA verified that nocodazole-induced mitosis block resulted in tetraploid cells. Rb and histone H3 were maximally hyperphosphorylated by 24 h of nocodazole treatment, accompanied by cell and nuclear size decreases and cellular rounding. Cells remained tetraploid and mononucleated with longer treatments, but other targets reverted to G1 levels, including Rb and ...

Real-Time and High Throughput Monitoring of cAMP in Live Cells Using a Fluorescent Membrane Potential-Sensitive Dye

Abstract: 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 serumfree environments. Upon stimulating the endogenous or heterogenous GPCRs on CNGC-cloned human embryonic kidney 293 cells with agonists, the fluorescence signal of HLB 021-15...

Validation of DRG-like F11 cells for evaluation of KCNQ/M-channel modulators.

Abstract: F11 cells are derived from a fusion between mouse embryonic neuroblastoma and rat dorsal root ganglion (DRG) neurons. These cells have been shown to retain many features of native DRG neurons, including synthesis of neurotransmitters, expression of neuropeptide receptors, and voltage-gated calcium channels. In this study, we describe the presence of KCNQ2/3 channels in F11 cells as determined by both reverse transcription-polymerase chain reaction and functional assessment. Electrophysiological recordings in whole-cell configuration performed in F11 cells revealed the functional expression of a KCNQ/M-current with characteristic slow deactivation kinetics, similar to the KCNQ/M-current recorded from dissociated DRG neurons. Deactivation tail currents elicited by conventional M-current protocols were enhanced by a specific KCNQ/M-channel opener, WAY-1, and inhibited by the specific blocker XE991 [10,10-bis(4-pyridinylmethyl)-9(10H)- anthracenone]. Using a non-radioactive atomic absorption Rb+ efflux assay,...

Profiling Activities of Transcription Factors in Breast Cancer Cell Lines

Abstract: Transcription factors (TFs) are critical regulators of cell growth and differentiation, whose dysfunction is associated with many human diseases, including cancer. To facilitate the discovery of functionally altered TFs among the approximately 2,000 human TFs, we (Panomics, Inc., Fremont, CA) developed a Protein/DNA array technology that can be used to profile the activities of multiple TFs simultaneously. In this study, we applied this technology to examine the TF activities in three different breast cancer cell lines: MCF7 (estrogen receptor [ER] +, tamoxifen-sensitive), T47D (ER+, tamoxifen-resistant), and HCC1806 (ER–, tamoxifen-resistant). We compared the differences in TF activity in these cells lines following treatment with estradiol or tamoxifen. We found a number of TF activities unique to each of these cell lines. In addition to verifying previous findings, the novel findings of this study provide a more comprehensive view of the differences in the response of these cancer lines to estrogen and...

Rb+ efflux through functional activation of cardiac KCNQ1/minK channels by the benzodiazepine R-L3 (L-364,373).

Abstract: The slow delayed rectifier K+ current, Iks, encoded by KCNQ1 (KvLQT1)/KCNE1 (mink) genes, contributes to cardiac action potential repolarization and determines the heartbeat rate. Mutations in either KCNQ1 or KCNE1 that reduce Iks cause long-QT syndrome (LQTS), a disorder of ventricular repolarization that results in cardiac arrhythmia and sudden death. A well-recognized potential treatment for LQTS caused by reduction of Iks is to enhance functional activation of cardiac KCNQ1/KCNE1 channels. In the present study, we generated a stable Chinese hamster ovary cell line that expresses KCNQ1/KCNE1 channels confirmed by electrophysiology. Using a pharmacological tool compound R-L3 (L-364,373 [(3-R)-1,3-dihydro-5-(2-fluorophenyl)-3-(1H-indol- 3-ylmethyl)-1-methyl-2H-1,4-benzodiazepin-2-one]), which activates KCNQ1/mink channels, we then developed and validated a non-radioactive rubidium (Rb+) efflux assay that directly measures the functional activity of KCNQ1/KCNE1 channels by atomic absorption spectroscopy. ...

Development of a fluorescent microsphere-based multiplexed high-throughput assay system for profiling of transcription factor activation.

Abstract: Transcription factors (TFs), which play crucial roles in the regulation of gene expression in the human genome, are highly regulated by a variety of mechanisms. A single extracellular stimulus can trigger multiple signaling pathways, and these in turn can activate multiple TFs to mediate the inducible expression of target genes. Alterations in the activities of TFs are often associated with human diseases, such as altered activating factor 1, estrogen receptor, and p53 function in cancer, nuclear factor κB in inflammatory diseases, and peroxisome proliferator-activated receptor γ in obesity. A systematic assay for profiling the activation of TFs will aid in elucidating the mechanisms of TF activation, reveal altered TFs associated with human diseases, and aid in developing assays for drug discovery. Here, we developed a 24-plex fluorescent microsphere-based TF activation assay system with a 96-well plate format. The assay system enabled high-throughput profiling of the DNA binding activity of TFs in multi...

Molecular beacons for DNA binding proteins: an emerging technology for detection of DNA binding proteins and their ligands.

Abstract: Quantitation of the level or activity of specific proteins is one of the most commonly performed experiments in biomedical research. Protein detection has historically been difficult to adapt to hi...

Real-time analysis of enzyme kinetics via micro parallel liquid chromatography.

Abstract: A generic method for real-time monitoring of enzyme kinetics is described in this paper. This approach enables rapid development of assays for high-throughput screening or reaction monitoring in the linear range of the enzyme kinetic curve. In this paper, we used protein kinase A and kemptide (a well-studied assay system) to demonstrate assay optimization by using micro parallel liquid chromatography. The optimal substrate and enzyme concentrations were determined rapidly and conveniently compared with the traditional methods for determining these parameters. Additionally, the data collected from the same experiment permitted calculations of K m for the substrate, V max, and time-course study. In general, this approach provides two advantages. First, the broad ranges of detectable product conversions facilitate selection and implementation of assay conditions for high-throughput screening. Second, the system permits determination of 50% inhibitory concentration values at less than 1% conversion of substra...

Investigating cytoskeletal alterations as a potential marker of retinal and lens drug-related toxicity.

Abstract: Actin filaments play a critical role in the normal physiology of lenticular and retinal cells in the eye. Disruption of the actin cytoskeleton has been associated with retinal pathology and lens cataract formation. Ocular toxicity is an infrequent observation in drug safety studies, yet its impact to the drug development process is significant. Recognizing compounds through screening with a potential ocular safety liability is one way to prioritize development candidates while reducing development attrition. Lens epithelial cells from human, dog, and rat origins and retinal pigmented epithelium cells from human, monkey, and rat origins were cultured and investigated with immunocytochemical techniques. Cells were treated using noncytotoxic doses of the compound, fixed, stained for actin with rhodamine phalloidin, and counterstained for nuclei with TOTO-3, followed by confocal imaging. Tamoxifen and several experimental compounds known to be in vivo lens and retinal toxicants caused a reduction in F-actin f...

A cell-sparing electric field stimulation technique for high-throughput screening of voltage-gated ion channels.

Abstract: The Trans Cell Layer Electrical Field Stimulation (TCL-EFS) system has been developed for high-throughput screening (HTS) of voltage-gated ion channels in microplate format on a Voltage-Ion Probe R...