High-throughput screening of drug-binding dynamics to HERG improves early drug safety assessment

2013 ◽  
Vol 304 (1) ◽  
pp. H104-H117 ◽  
Author(s):  
Giovanni Y. Di Veroli ◽  
Mark R. Davies ◽  
Henggui Zhang ◽  
Najah Abi-Gerges ◽  
Mark R. Boyett
Keyword(s):  
High Throughput ◽  
Drug Safety ◽  
High Throughput Screening ◽  
Chinese Hamster Ovary Cells ◽  
Torsade De Pointes ◽  
Chinese Hamster ◽  
Drug Binding ◽  
Binding Kinetics ◽  
Quantitative Prediction ◽  
Delayed Rectifier

The use of computational models to predict drug-induced changes in the action potential (AP) is a promising approach to reduce drug safety attrition but requires a better representation of more complex drug-target interactions to improve the quantitative prediction. The blockade of the human ether-a-go-go-related gene (HERG) channel is a major concern for QT prolongation and Torsade de Pointes risk. We aim to develop quantitative in-silico AP predictions based on a new electrophysiological protocol (suitable for high-throughput HERG screening) and mathematical modeling of ionic currents. Electrophysiological recordings using the IonWorks device were made from HERG channels stably expressed in Chinese hamster ovary cells. A new protocol that delineates inhibition over time was applied to assess dofetilide, cisapride, and almokalant effects. Dynamic effects displayed distinct profiles for these drugs compared with concentration-effects curves. Binding kinetics to specific states were identified using a new HERG Markov model. The model was then modified to represent the canine rapid delayed rectifier K+ current at 37°C and carry out AP predictions. Predictions were compared with a simpler model based on conductance reduction and were found to be much closer to experimental data. Improved sensitivity to concentration and pacing frequency variables was obtained when including binding kinetics. Our new electrophysiological protocol is suitable for high-throughput screening and is able to distinguish drug-binding kinetics. The association of this protocol with our modeling approach indicates that quantitative predictions of AP modulation can be obtained, which is a significant improvement compared with traditional conductance reduction methods.

scholarly journals Development of a Ca2+-Activated Photoprotein, Photina®, and Its Application to High-Throughput Screening

2007 ◽  
Vol 12 (5) ◽  
pp. 694-704 ◽  
Author(s):  
Silvia Bovolenta ◽  
Maria Foti ◽  
Stefan Lohmer ◽  
Sabrina Corazza
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Signal To Noise Ratio ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Cell Number ◽  
Ovary Cells ◽  
Mammalian Cell Lines ◽  
Calcium Dyes ◽  
G Protein Coupled

The present work describes the engineering and characterization of a new Ca2+-activated photoprotein (Photina®) and its use in mammalian cell lines for implementation of flash luminescence cell-based assays for high-throughput screening (HTS). When used to measure the activation of 2 G protein—coupled receptors (GPCRs), targeting Photina® to the mitochondria increased the signal strength as compared to the normal cytoplasmic expression of Photina.® The mitochondrial-targeted Photina® also produced a higher signal-to-noise ratio than conventional calcium dyes and a consistently stronger signal than aequorin when tested under equivalent conditions. MitoPhotina® provided strong and reliable results when used to measure the activity of purinergic receptors endogenously expressed in the Chinese Hamster Ovary cells and heterologously expressed GPCRs in response to their cognate ligands. Several different types of flash luminescence plate readers (FLIPR3, FLIPRTETRA, CyBi®-®Lumax flash HT, Lumilux®, Lumibox) in different plate formats (96, 384, 1536 wells) were used to validate the use of Photina in HTS. The cell number had to be adjusted to correspond to the qualities of the different readers, but once so adjusted, it provided equivalent results on each device. The results obtained show robust and reproducible light signals that offer new possibilities for application of photoproteins to the generation of cell-based assays for HTS. ( Journal of Biomolecular Screening 2007:694-704)

High-Throughput Screening for Ligand-Induced c-fos mRNA Expression by Branched DNA Assay in Chinese Hamster Ovary Cells

1999 ◽  
Vol 266 (1) ◽  
pp. 140-147 ◽  
Author(s):  
Venkatakrishna Shyamala ◽  
Hamiduddin Khoja ◽  
Mary L. Anderson ◽  
Jian-xin Wang ◽  
Hui Cen ◽  
...  
Keyword(s):  
Mrna Expression ◽  
High Throughput ◽  
Chinese Hamster Ovary ◽  
High Throughput Screening ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Ovary Cells ◽  
Dna Assay ◽  
Branched Dna

scholarly journals Development of a Whole Cell High Throughput Screen for Muscarinic Receptor Agonists

1997 ◽  
Vol 2 (2) ◽  
pp. 79-84 ◽  
Author(s):  
Stuart W. Bright ◽  
Jill D. Higginbotham ◽  
Divann J. Cofield ◽  
Julie F. Falcone ◽  
Frank P. Bymaster
Keyword(s):  
Muscarinic Receptor ◽  
High Throughput ◽  
Chinese Hamster Ovary Cells ◽  
Muscarinic Acetylcholine Receptor ◽  
Chinese Hamster ◽  
Growth Media ◽  
High Throughput Screen ◽  
Muscarinic Agonists ◽  
Intact Cells ◽  
Receptor Agonists

We have developed a high throughput screen using intact cells to identify muscarinic receptor agonists. Chinese hamster ovary cells stably transfected with the Ml muscarinic acetylcholine receptor (CHO-Ml) were pre-labeled with [3HJ-arachidonic acid (AA). Stimulation of muscarinic receptors with known muscarinic agonists resulted in release of AA from the cells into the culture medium. The released [3H]-AA in this assay was counted using both standard scintillation methods and Luma Plates. Because muscarinic antagonists do not cause release of AA, only agonists are identified. A follow-up screen using a competitive antagonist was used to confirm agonist properties of active compounds. This screen was relatively simple, reproducible, and compatible with many organic solvents and natural products growth media. Thus, it may be useful for the discovery of muscarinic agonists from natural product broths or synthetic compounds.

Development of a β-Lactamase Reporter Gene Assay for Metabotropic Glutamate Receptor 1 by Using Coexpression of Glutamate Transporter

2010 ◽  
Vol 15 (2) ◽  
pp. 148-158 ◽  
Author(s):  
Gentaroh Suzuki ◽  
Hiroshi Kawamoto ◽  
Hisashi Ohta
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Metabotropic Glutamate Receptor ◽  
Chinese Hamster ◽  
High Sensitivity ◽  
Reporter Assay ◽  
Activated T Cells ◽  
Basal Activity ◽  
Gene Assay ◽  
Very High

mGluR1 antagonists have been postulated to be novel CNS drugs, including antipsychotics. Toward this end, the authors developed a β-lactamase reporter assay to identify mGluR1 antagonists. β-Lactamase has several interesting features for high-throughput screening, including very high sensitivity and less well-to-well variation than other reporter enzymes. mGluR1-expressing Chinese hamster ovary (CHO) cells with the β-lactamase gene under control of the nuclear factor of activated T cells (NFAT) promoter (CHO-NFAT-bla-hmGluR1b) exhibited very high basal activity, resulting in an inadequate signal-to-basal (S/B) ratio. Coexpression of glutamate/aspartate transporter (GLAST) with mGluR1 in the cell line (CHO-NFAT-bla-hmGluR1b-GLAST) dramatically decreased basal activity and improved the S/B ratio (from 2- to 20-fold). The contribution of GLAST to lowering basal activity and increasing the S/B ratio was validated by the expression level of GLAST mRNA and by a GLAST inhibitor. Antagonistic activities of known mGluR1 antagonists in the β-lactamase reporter assay were comparable with those in the conventional Ca2+ mobilization assay. The Z′ factor of the β-lactamase reporter assay was 0.89 under optimized conditions. Taken together, the β-lactamase reporter assay with CHO-NFAT-bla-hmGluR1b-GLAST could be a novel high-throughput assay for mGluR1 antagonist screening. This is the first description of a successful β-lactamase reporter assay among all mGluR subtypes.

scholarly journals Fluorescence Polarization Assays for High Throughput Screening of G Protein-Coupled Receptors

2000 ◽  
Vol 5 (3) ◽  
pp. 159-167 ◽  
Author(s):  
Peter Banks ◽  
Mylene Gosselin ◽  
Linda Prystay
Keyword(s):  
High Throughput ◽  
Fluorescence Polarization ◽  
High Throughput Screening ◽  
Rank Order ◽  
Drug Binding ◽  
G Protein Coupled Receptors ◽  
Microtiter Plates ◽  
High Throughput Drug Screening ◽  
G Protein Coupled ◽  
Speed Of Analysis

Fluorescence polarization assays in 384-well microtiter plates have been demonstrated. The performance is suitable for high throughput drug screening applications with respect to speed of analysis, displaceable signal, precision, and sensitivity to various reagents. Rank order of potency was maintained relative to ['251]-ligand filtration assays, and the effects of the highly colored compounds, tartrazine and Chicago Sky Blue, were insignificant on the polarization signal up to a concentration of 1 tiM. These attributes suggest that accurate assessment of drug binding can be obtained.

scholarly journals A Cell-Based Ultra-High-Throughput Screening Assay for Identifying Inhibitors of D-Amino Acid Oxidase

2006 ◽  
Vol 11 (5) ◽  
pp. 481-487 ◽  
Author(s):  
Philip E. Brandish ◽  
Chi-Sung Chiu ◽  
Jonathan Schneeweis ◽  
Nicholas J. Brandon ◽  
Clare L. Leech ◽  
...  
Keyword(s):  
Amino Acid ◽  
High Throughput ◽  
High Throughput Screening ◽  
Hormone Receptors ◽  
Chinese Hamster ◽  
Acid Oxidase ◽  
Amino Acid Oxidase ◽  
Compound Libraries ◽  
High Throughput Screening Assay ◽  
Artificial Environment

Enzymes are often considered less “druggable” targets than ligand-regulated proteins such as G-protein-coupled receptors, ion channels, or other hormone receptors. Reasons for this include cellular location (intracellular vs. cell surface), typically lower affinities for the binding of small molecules compared to ligand-specific receptors, and binding (catalytic) sites that are often charged or highly polar. A practical drawback to the discovery of compounds targeting enzymes is that screening of compound libraries is typically carried out in cell-free activity assays using purified protein in an inherently artificial environment. Cell-based assays, although often arduous to design for enzyme targets, are the preferred discovery tool for the screening of large compound libraries. The authors have recently described a novel cell-based approach to screening for inhibitors of a phosphatase enzyme and now report on the development and implementation of a homogeneous 3456-well plate assay for D-amino acid oxidase (DAO). Human DAO was stably expressed in Chinese hamster ovary (CHO) cells, and its activity was measured as the amount of hydrogen peroxide detected in the growth medium following feeding the cells with D-serine. In less than 12 weeks, the authors proved the concept in 96-and then 384-well formats, miniaturized the assay to the 3456-well (nanoplate) scale, and screened a library containing more than 1 million compounds. They have identified several cell-permeable inhibitors of DAO from this cell-based high-throughput screening, which provided the discovery program with a few novel and attractive lead structures.

Circular dichroism and synchrotron radiation circular dichroism spectroscopy: tools for drug discovery

2003 ◽  
Vol 31 (3) ◽  
pp. 631-633 ◽  
Author(s):  
B.A. Wallace ◽  
Robert W. Janes
Keyword(s):  
Circular Dichroism ◽  
Drug Discovery ◽  
Synchrotron Radiation ◽  
High Throughput ◽  
Conformational Changes ◽  
High Throughput Screening ◽  
Cd Spectroscopy ◽  
Drug Binding ◽  
Apoptosis Protein ◽  
Circular Dichroïsm

CD spectroscopy is an established and valuable technique for examining protein structure, dynamics and folding. Because of its ability to sensitively detect conformational changes, it has important potential for drug discovery, enabling screening for ligand and drug binding, and detection of potential candidates for new pharmaceuticals. The binding of the anti-tumour agent Taxol to the anti-apoptosis protein Bcl-2 [Rodi, Janes, Sanganee, Holton, Wallace and Makowski (1999) J. Mol. Biol. 285, 197–204] and the binding of the anti-epileptic drug lamotrigine to voltage-gated sodium channels [Cronin, O'Reilly, Duclohier and Wallace (2003) J. Biol. Chem. 278, 10675–10682] are used as examples to show changes detectable by CD involving secondary structure, and are contrasted with the binding of the agonist carbamylcholine to acetylcholine receptors [Mielke and Wallace (1988) J. Biol. Chem. 263, 8177–8182], an example where binding does not involve a secondary structural change. Synchrotron radiation CD spectroscopy offers significant enhancements with respect to conventional CD spectroscopy, which will enable its usage for high-throughput screening and as a tool in ‘chemical genomics’ or ‘reverse chemical genetics’ strategies for ligand identification. The lower wavelength data available enable more detailed, sensitive and accurate detection, the higher light intensity permits much smaller amounts of both proteins and drug candidates to be used in the screening, and future technological developments in sample handling and detection should enable automated high-throughput screening to be performed.

scholarly journals High throughput, efficacious gene editing & genome surveillance in Chinese hamster ovary cells

PLoS ONE ◽  
2019 ◽  
Vol 14 (12) ◽  
pp. e0218653 ◽  
Author(s):  
S. C. Huhn ◽  
Y. Ou ◽  
A. Kumar ◽  
R. Liu ◽  
Z. Du
Keyword(s):  
High Throughput ◽  
Chinese Hamster Ovary ◽  
Gene Editing ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Ovary Cells

scholarly journals Effect of channel assembly (KCNQ1 or KCNQ1 + KCNE1) on the response of zebrafish IKs to IKs inhibitors and activators

2021 ◽  
Author(s):  
Jaakko Haverinen ◽  
Minna Hassinen ◽  
Matti Vornanen
Keyword(s):  
Mefenamic Acid ◽  
Slow Component ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Delayed Rectifier ◽  
Model Species ◽  
Ovary Cells ◽  
Key Factor ◽  
Channel Assembly ◽  
Adrenergic Activation

ABSTRACTIn cardiac myocytes, the slow component of the delayed rectifier K+ current (IKs) ensures repolarization of action potential during beta-adrenergic activation or when other repolarizing K+ currents fail. As a key factor of cardiac repolarization IKs should be present in model species used for cardiovascular drug screening, preferably with pharmacological characteristics similar to those of the human IKs. To this end, we investigated the effects of inhibitors and activators of the IKs on KCNQ1 and KCNQ1+KCNE1 channels of the zebrafish, an important model species, in Chinese hamster ovary cells. Inhibitors of IKs, chromanol 293B and HMR-1556, inhibited zebrafish IKs channels with approximately similar potency as that of mammalian IKs. Chromanol 293B concentration for half-maximal inhibition (IC50) of zebrafish IKs was at 13.1±5.8 and 13.4±2.8 μM for KCNQ1 and KCNQ1+KCNE1 channels, respectively. HMR-1556 was a more potent inhibitor of zebrafish IKs with IC50=0.1±0.1 μM and 1.5±0.8 μM for KCNQ1 and KCNQ1+KCNE1 channels, respectively. R-L3 and mefenamic acid, generally identified as IKs activators, both inhibited zebrafish IKs. R-L3 almost completely inhibited zebrafish IKs generated by KCNQ1 and KCNQ1+KCNE1 channels with similar affinity (IC50 1.1±0.4 and 1.0±0.4 μM, respectively). Mefenamic acid partially blocked zebrafish KCNQ1 (IC50=9.5±4.8 μM) and completely blocked KCNQ1+KCNE1 channels (IC50=3.3±1.8 μM). Although zebrafish IKs responds to IKs inhibitors in the same way as mammalian IKs, its response to activators is atypical, probably due to the differences in the binding domain of KCNE1 to KCNQ1. Therefore, care must be taken when translating the results from zebrafish to humans.

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