Book contents
- Frontmatter
- Contents
- Contributors
- Preface
- Section one Overviews
- Section two Molecules for Chemical Genomics
- Section Three Basics of High-Throughput Screening
- Section Four Chemical Genomics Assays and Screens
- Chapter 12 Basics of HTS Assay Design and Optimization
- Chapter 13 Molecular Sensors for Transcriptional and Post-Transcriptional Assays
- Chapter 14 Time-Resolved Fluorescence Resonance Energy Transfer Technologies in HTS
- Chapter 15 Compound Profiling with High-Content Screening Methodology
- Chapter 16 Use of Transgenic Zebrafish in a Phenotypic Screen for Angiogenesis Inhibitors
- Chapter 17 Flow Cytometry Multiplexed Screening Methodologies
- Chapter 18 Label-Free Biosensor Technologies in Small Molecule Modulator Discovery
- Chapter 19 Basic Principles and Practices of Computer-Aided Drug Design
- Chapter 20 Computational Approach for Drug Target Identification
- Section five Chemical Genomics and Medicine
- Index
- References
Chapter 13 - Molecular Sensors for Transcriptional and Post-Transcriptional Assays
from Section Four - Chemical Genomics Assays and Screens
Published online by Cambridge University Press: 05 June 2012
Edited by
Book contents
- Frontmatter
- Contents
- Contributors
- Preface
- Section one Overviews
- Section two Molecules for Chemical Genomics
- Section Three Basics of High-Throughput Screening
- Section Four Chemical Genomics Assays and Screens
- Chapter 12 Basics of HTS Assay Design and Optimization
- Chapter 13 Molecular Sensors for Transcriptional and Post-Transcriptional Assays
- Chapter 14 Time-Resolved Fluorescence Resonance Energy Transfer Technologies in HTS
- Chapter 15 Compound Profiling with High-Content Screening Methodology
- Chapter 16 Use of Transgenic Zebrafish in a Phenotypic Screen for Angiogenesis Inhibitors
- Chapter 17 Flow Cytometry Multiplexed Screening Methodologies
- Chapter 18 Label-Free Biosensor Technologies in Small Molecule Modulator Discovery
- Chapter 19 Basic Principles and Practices of Computer-Aided Drug Design
- Chapter 20 Computational Approach for Drug Target Identification
- Section five Chemical Genomics and Medicine
- Index
- References
Summary
Cell-based assays allow researchers to probe the regulation of cell signaling, communication, and regulation within a system that is more physiologically relevant than assays that focus on isolated molecular components or cell extracts (biochemical assays). The application of cell-based assays in high-throughput screening (HTS) has increased in recent years. It is estimated that cell-based assays comprise more than half of HTS assays used in pharmaceutical industries [1].
Cell-based molecular sensor assays have been developed to measure either transcriptional or post-transcriptional events (Figure 13.1). A typical transcriptional reporter gene assay involves the placement of a cis-regulatory element in front of a gene that produces a measurable signal in response to modulation of transcriptional complexes that are regulated by ligands or complex networks. Assays favored in HTS are usually constructed to generate fluorescence or luminescence by expressing either an enzyme or a fluorescent protein reporter. Post-transcriptional assays have been developed by engineering the reporter to respond to events such as mRNA splicing, protein complex formation, translocation of a receptor between cellular compartments, or protein stability.
- Type
- Chapter
- Information
- Chemical Genomics , pp. 173 - 197Publisher: Cambridge University PressPrint publication year: 2012
References
2007 High-throughput screening assays for the identification of chemical probesNat Chem Biol 3 466Google Scholar
1980 The structure of beta-lactamasesPhilosophical transactions of the Royal Society of London 289 321Google Scholar
2000 Fusions to beta-lactamase as a reporter for gene expression in live mammalian cellsMethods Enzymol 326 221Google Scholar
1978 Nucleotide sequence of the ampicillin resistance gene of plasmid pBR322Proc Natl Acad Sci U S A 75 3737Google Scholar
1998 Quantitation of transcription and clonal selection of single living cells with beta-lactamase as reporterScience 279 84Google Scholar
2005 High-throughput screening using beta-lactamase reporter-gene technology for identification of low-molecular-weight antagonists of the human gonadotropin releasing hormone receptorAssay Drug Dev Technol 3 143Google Scholar
2003 Development of an intact cell reporter gene beta-lactamase assay for G protein-coupled receptors for high-throughput screeningAnal Biochem 314 16Google Scholar
2003 Detection of beta-lactamase reporter gene expression by flow cytometryCytometry A 51 68Google Scholar
2006 Application of Laser-Scanning Fluorescence Microplate Cytometry in High Content ScreeningAssay Drug Dev Technol 4 209Google Scholar
2003 Miniaturization of cell-based beta-lactamase-dependent FRET assays to ultra-high throughput formats to identify agonists of human liver X receptorsAssay Drug Dev Technol 1 777Google Scholar
2003 A beta-lactamase-dependent Gal4-estrogen receptor beta transactivation assay for the ultra-high throughput screening of estrogen receptor beta agonists in a 3456-well formatAssay Drug Dev Technol 1 789Google Scholar
2007 An improved beta-lactamase reporter assay: multiplexing with a cytotoxicity readout for enhanced accuracy of hit identificationJ Biomol Screen 12 635Google Scholar
2006 A comparison of assay performance measures in screening assays: signal window, Zʹ factor, and assay variability ratioJ Biomol Screen 11 247Google Scholar
1999 A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening AssaysJ Biomol Screen 4 67Google Scholar
2004 A cell-based beta-lactamase reporter gene assay for the identification of inhibitors of hepatitis C virus replicationAnal Biochem 334 344Google Scholar
2003 Identification of signal transduction pathways used by orphan G protein-coupled receptorsAssay and Drug Development Technologies 1 239Google Scholar
http://pubchem.ncbi.nlm.nih.gov/
2006 Quantitative high-throughput screening: A titration-based approach that efficiently identifies biological activities in large chemical librariesProc Natl Acad Sci U S A 103 11473Google Scholar
2010 Apparent activity in high-throughput screening: origins of compound-dependent assay interferenceCurr Opin Chem Biol 14 315Google Scholar
2008 Comprehensive Mechanistic Analysis of Hits from High-Throughput and Docking Screens against beta-LactamaseJ Med Chem 51 2502Google Scholar
2002 A comparison of ALPHAScreen, TR-FRET, and TRF as assay methods for FXR nuclear receptorsJ Biomol Screen 7 3Google Scholar
1962 Extraction, purification and properties of aequorin, a bioluminescent protein from the luminous hydromedusan, AequoreaJ Cell Comp Physiol 59 223Google Scholar
1993 Chemical structure of the hexapeptide chromophore of the Aequorea green-fluorescent proteinBiochemistry 32 1212Google Scholar
1962 Quantum efficiency of Cypridina luminescence with a note on that of AequoreaJ Cell Comp Physiol 60 85Google Scholar
1995 Understanding, improving and using green fluorescent proteinsTrends Biochem Sci 20 448Google Scholar
2001 The color of mice: in the light of GFP-variant reportersHistochemistry and cell biology 115 49Google Scholar
1996 Engineering green fluorescent protein for improved brightness, longer wavelengths and fluorescence resonance energy transferCurr Biol 6 178Google Scholar
2000 Biochemistry, mutagenesis, and oligomerization of DsRed, a red fluorescent protein from coralProc Natl Acad Sci U S A 97 11984Google Scholar
2006 Cyan and yellow super fluorescent proteins with improved brightness, protein folding, and FRET Forster radiusBiochemistry 45 6570Google Scholar
2008 Improving the photostability of bright monomeric orange and red fluorescent proteinsNat Methods 5 545Google Scholar
2007 Recent advances using green and red fluorescent protein variantsApplied microbiology and biotechnology 77 1Google Scholar
1995 Quantitative imaging of green fluorescent protein in cultured cells: comparison of microscopic techniques, use in fusion proteins and detection limitsJournal of microscopy 180 109Google Scholar
1999 Attenuation of green fluorescent protein half-life in mammalian cellsProtein engineering 12 1035Google Scholar
1986 Amino acid sequences common to rapidly degraded proteins: the PEST hypothesisScience 234 364Google Scholar
1998 Generation of destabilized green fluorescent protein as a transcription reporterJ Biol Chem 273 34970Google Scholar
2007 High throughput drug screening for human immunodeficiency virus type 1 reactivating compoundsAssay Drug Dev Technol 5 181Google Scholar
2006 T-cell line for HIV drug screening using EGFP as a quantitative marker of HIV-1 replicationBiotechniques 40 91Google Scholar
2003 Generation of stably transfected Mammalian cell lines as fluorescent screening assay for NF-kappaB activation-dependent gene expressionJ Biomol Screen 8 511Google Scholar
2006 Measuring Biological Responses with Automated MicroscopyElsevier Academic PressSan Diego
2006 Fluorescent protein-based cellular assays analyzed by laser-scanning microplate cytometry in 1536-well plate formatMethods Enzymol 414 566Google Scholar
1985 Cloning of firefly luciferase cDNA and the expression of active luciferase in Escherichia coliProc Natl Acad Sci U S A 82 7870Google Scholar
1987 Firefly luciferase gene: structure and expression in mammalian cellsMol Cell Biol 7 725Google Scholar
1991 Modulation of firefly luciferase stability and impact on studies of gene regulationGene 103 171Google Scholar
2000 Development of a destabilized firefly luciferase enzyme for measurement of gene expressionBiotechniques 29 590594Google Scholar
2008 A chemical biology approach reveals period shortening of the mammalian circadian clock by specific inhibition of GSK-3betaProc Natl Acad Sci U S A 105 20746Google Scholar
1991 Membrane-permeable luciferin esters for assay of firefly luciferase in live intact cellsBiochem J 276 637Google Scholar
2004 Kinetic analysis and modeling of firefly luciferase as a quantitative reporter gene in live mammalian cellsBiotechnol Bioeng 86 827Google Scholar
2002 Imaging of light emission from the expression of luciferases in living cells and organisms: a reviewLuminescence 17 43Google Scholar
1978 Mechanism of the enzyme-catalyzed bioluminescent oxidation of coelenterate-type luciferinBiochem Biophys Res Commun 81 980Google Scholar
1978 Energy transfer via protein-protein interaction in bioluminescencePhotochemistry and Photobiology 27 389Google Scholar
2006 Consensus guided mutagenesis of luciferase yields enhanced stability and light outputProtein Eng Des Sel 19 391Google Scholar
1998 Rapid luciferase reporter assay systems for high throughput studiesPromega Notes 65 9Google Scholar
1999 Development of a dual glow-signal firefly and Renilla luciferase assay reagent for the analysis of G-protein coupled receptor signallingJ Recept Signal Transduct Res 19 395Google Scholar
2007 9
2000 A noncommercial dual luciferase enzyme assay system for reporter gene analysisAnal Biochem 282 158Google Scholar
2006 A protocol for combined and luciferase quantification compatible with protein assaysAnal Biochem 356 94Google Scholar
1989 Complementary DNA coding click beetle luciferases can elicit bioluminescence of different colorsScience 244 700Google Scholar
2007 A cell-based assay for IkappaBalpha stabilization using A two-color dual luciferase-based sensorAssay Drug Dev Technol 5 85Google Scholar
2008 Characterization of chemical libraries for luciferase inhibitory activityJ Med Chem 51 2372Google Scholar
2008 A specific mechanism for nonspecific activation in reporter-gene assaysACS Chem Biol 3 463Google Scholar
2010 Illuminating insights into firefly luciferase and other bioluminescent reporters used in chemical biologyChem Biol 17 646Google Scholar
1996 Crystal structure of firefly luciferase throws light on a superfamily of adenylate-forming enzymesStructure 4 287Google Scholar
2006 Structural basis for the spectral difference in luciferase bioluminescenceNature 440 372Google Scholar
1997 Mutation of a protease-sensitive region in firefly luciferase alters light emission propertiesJ Biol Chem 272 18766Google Scholar
2001 Luminescent proteins from Aequorea victoria: applications in drug discovery and in high throughput analysisFresenius J Anal Chem 369 258Google Scholar
1998 Trends Biotechnol 16 216
1995 Transfected aequorin in the measurement of cytosolic Ca2+ concentration ([Ca2+]c). A critical evaluationJ Biol Chem 270 9896Google Scholar
1985 Cloning and sequence analysis of cDNA for the luminescent protein aequorinProc Natl Acad Sci U S A 82 3154Google Scholar
1999 Targeted recombinant aequorins: tools for monitoring [Ca2+] in the various compartments of a living cellMicroscopy research and technique 46 380Google Scholar
1985 Cloning and expression of the cDNA coding for aequorin, a bioluminescent calcium-binding proteinBiochem Biophys Res Commun 126 1259Google Scholar
1993 Measurement of intracellular calcium using bioluminescent aequorin expressed in human cellsAnal Biochem 209 343Google Scholar
1990 Photoprotein aequorin: use as a reporter enzyme in studying gene expression in mammalian cellsGene 96 249Google Scholar
2002 Adaptation of aequorin functional assay to high throughput screeningJournal of Biomolecular Screening 7 57Google Scholar
2007 Development of a Ca2+-activated photoprotein, Photina, and its application to high-throughput screeningJ Biomol Screen 12 694Google Scholar
2000 A high-throughput glow-type aequorin assay for measuring receptor-mediated changes in intracellular calcium levelsAnal Biochem 286 231Google Scholar
1993 The relative rate of aequorin regeneration from apoaequorin and coelenterazine analoguesBiochem J 296 549Google Scholar
2000 Uses of lacZ to study gene function: evaluation of beta-galactosidase assays employed in the yeast two-hybrid systemAnal Biochem 285 1Google Scholar
2003 Reporter gene assays and their applications to bioassays of natural productsPhytother Res 17 439Google Scholar
1990 Reporter genes: application to the study of mammalian gene transcriptionAnal Biochem 188 245Google Scholar
1996 Dual luminescence-based reporter gene assay for luciferase and beta-galactosidaseBiotechniques 21 520Google Scholar
2003 Beta galactosidase enzyme fragment complementation as a novel technology for high throughput screeningComb Chem High Throughput Screen 6 381Google Scholar
1988 Secreted placental alkaline phosphatase: a powerful new quantitative indicator of gene expression in eukaryotic cellsGene 66 1Google Scholar
1991 A pitfall of using a second plasmid to determine transfection efficiencyNucleic Acids Research 20 920Google Scholar
2005 Inhibitory effects associated with use of modified and luciferase vectors in dual reporter assays and implications for analysis of ISGsJ Interferon Cytokine Res 25 92Google Scholar
1996 Induction of NFAT-mediated transcription by Gq-coupled receptors in lymphoid and non-lymphoid cellsJ Biol Chem 271 10429Google Scholar
2001 Reporter-gene systems for the study of G-protein-coupled receptorsCurr Opin Pharmacol 1 526Google Scholar
2000 Gene expression elicited by NFAT in the presence or absence of cooperative recruitment of Fos and JunEMBO J 19 4783Google Scholar
2003 Steroid hormone receptors and drug discovery: therapeutic opportunities and assay designsAssay Drug Dev Technol 1 843Google Scholar
2003 Coactivators in assay design for nuclear hormone receptor drug discoveryAssay Drug Dev Technol 1 835Google Scholar
2003 Multiplexing nuclear receptors for agonist identification in a cell-based reporter gene high-throughput screenJ Biomol Screen 8 239Google Scholar
1995 Differential activation of c-fos promoter elements by serum, lysophosphatidic acid, G proteins and polypeptide growth factorsEMBO J 14 5037Google Scholar
1994 Signal transduction and gene regulation: the nuclear response to cAMPJ Biol Chem 269 17359Google Scholar
1996 Integration of growth factor signals at the c-fos serum response elementPhilosophical transactions of the Royal Society of London 351 551Google Scholar
2004 The IKK NF-kappa B system: a treasure trove for drug developmentNat Rev Drug Discov 3 17Google Scholar
1999 Characterization of NFkappaB activation by detection of green fluorescent protein-tagged IkappaB degradation in living cellsJ Biol Chem 274 21244Google Scholar
2005 Real-time imaging of ligand-induced IKK activation in intact cells and in living miceNat Methods 2 607Google Scholar
2000 A ubiquitin-based tagging system for controlled modulation of protein stabilityNature Biotechnology 18 1298Google Scholar
2006 Enzyme fragment complementation binding assay for p38alpha mitogen-activated protein kinase to study the binding kinetics of enzyme inhibitorsAssay Drug Dev Technol 4 411Google Scholar
2002 Enzyme fragment complementation: a flexible high throughput screening assay technologyAssay Drug Dev Technol 1 97Google Scholar
2007 Application of beta-lactamase enzyme complementation to the high-throughput screening of toll-like receptor signaling inhibitorsMol Pharmacol 72 868Google Scholar
2008 High-throughput cellular assays for regulated posttranslational modificationsAnal Biochem 372 189Google Scholar
2007 Universal strategies in research and drug discovery based on protein-fragment complementation assaysNat Rev Drug Discov 6 569Google Scholar
2006 Identifying off-target effects and hidden phenotypes of drugs in human cellsNat Chem Biol 2 329Google Scholar
2003 Protein fragment complementation strategies for biochemical network mappingCurr Opin Biotechnol 14 610Google Scholar
2004 Mapping biochemical networks with protein-fragment complementation assaysMethods Mol Biol 261 411Google Scholar
2006 The fluorescent toolbox for assessing protein location and functionScience 312 217Google Scholar
2008 The genetic design of signaling cascades to record receptor activationProc Natl Acad Sci U S A 105 64Google Scholar
2003 Measuring drug action in the cellular context using protein-fragment complementation assaysAssay Drug Dev Technol 1 811Google Scholar
2007 Quantification of dynamic protein complexes using luciferase fragment complementation applied to protein kinase A activities in vivoProc Natl Acad Sci U S A 104 16916Google Scholar
2006 A miniaturized cell-based fluorescence resonance energy transfer assay for insulin-receptor activationAnal Biochem 355 267Google Scholar
2006 Illuminating insights into protein-protein interactions using bioluminescence resonance energy transfer (BRET)Nat Methods 3 165Google Scholar
2007 J Mol Biol 374 1017
2002 The use of resonance energy transfer in high-throughput screening: BRET versus FRETTrends Pharmacol Sci 23 351Google Scholar
2005 High-throughput screening of G protein-coupled receptor antagonists using a bioluminescence resonance energy transfer 1-based beta-arrestin2 recruitment assayJ Biomol Screen 10 463Google Scholar
2007 Bioluminescent indicator for determining protein-protein interactions using intramolecular complementation of split click beetle luciferaseAnal Chem 79 4820Google Scholar
2006 High Content ScreeningTotowa, N.JHumana Press
2006 A homogeneous cell-based assay to measure nuclear translocation using beta-galactosidase enzyme fragment complementationAssay Drug Dev Technol 4 263Google Scholar
2009 A combination of ultrahigh throughput PathHunter and cytokine secretion assays to identify glucocorticoid receptor agonistsAnal Biochem 385 286Google Scholar
1998 All four homochiral enantiomers of a nuclear localization sequence derived from c-Myc serve as functional import signalsJ Biol Chem 273 29764Google Scholar
2008 A miniaturized glucocorticoid receptor translocation assay using enzymatic fragment complementation evaluated with qHTSComb Chem High Throughput Screen 11 545Google Scholar
2003 Measuring intracellular calcium fluxes in high throughput modeComb Chem High Throughput Screen 6 355Google Scholar
2004 cAMP detection methods in HTS: selecting the best from the restNat Rev Drug Discov 3 125Google Scholar
2001 Homogeneous cell-based fluorescence polarization assay for the direct detection of cAMPJ Biomol Screen 6 75Google Scholar
2003 High throughput screening technologies for direct cyclic AMP measurementAssay Drug Dev Technol 1 291Google Scholar
2004 A 1536-well cAMP assay for Gs- and Gi-coupled receptors using enzyme fragmentation complementationAssay Drug Dev Technol 2 39Google Scholar
1988 Calcium binding kinetics of fura-2 and azo-1 from temperature-jump relaxation measurementsBiophysical Journal 53 635Google Scholar
2008
1999 Measurement of [Ca2+] using the Fluorometric Imaging Plate Reader (FLIPR)Methods Mol Biol 114 125Google Scholar
2001 Is Galpha16 the optimal tool for fishing ligands of orphan G-protein-coupled receptorsTrends Pharmacol Sci 22 560Google Scholar
2003 Galpha(16/z) chimeras efficiently link a wide range of G protein-coupled receptors to calcium mobilizationJ Biomol Screen 8 39Google Scholar
2003 High-throughput real-time monitoring of Gs-coupled receptor activation in intact cells using cyclic nucleotide-gated channelsEur J Pharmacol 478 27Google Scholar
2001 Validation of FLIPR membrane potential dye for high throughput screening of potassium channel modulatorsJ Biomol Screen 6 305Google Scholar
2004 High throughput assay technologies for ion channel drug discoveryAssay Drug Dev Technol 2 543Google Scholar
2008 Quantitative high-throughput screening using a live-cell cAMP assay identifies small-molecule agonists of the TSH receptorJ Biomol Screen 13 120Google Scholar
2000 A genetically encoded, fluorescent indicator for cyclic AMP in living cellsNature Cell Biology 2 25Google Scholar
2002 Visualization of interactions among bZIP and Rel family proteins in living cells using bimolecular fluorescence complementationMol Cell 9 789Google Scholar
1997 Fluorescent indicators for Ca2+ based on green fluorescent proteins and calmodulinNature 388 882Google Scholar
2000 A fluorescent indicator for visualizing cAMP-induced phosphorylation in vivoNat Biotechnol 18 313Google Scholar
2007 Design of fluorescence resonance energy transfer (FRET)-based cGMP indicators: a systematic approachBiochem J 407 69Google Scholar
2004 Fluorescence properties of green fluorescent protein FRET pairs concatenated with the small G protein, Rac, and its interacting domain of the kinase, p21-activated kinaseAssay Drug Dev Technol 2 659Google Scholar
2000 Development and application of a GFP-FRET intracellular caspase assay for drug screeningJ Biomol Screen 5 307Google Scholar
2003 Genome-wide survey of human alternative pre-mRNA splicing with exon junction microarraysScience 302 2141Google Scholar
2006 Use of transcriptional synergy to augment sensitivity of a splicing reporter assayRNA 12 925Google Scholar
2008 A high-throughput screening strategy identifies cardiotonic steroids as alternative splicing modulatorsProc Natl Acad Sci U S A 105 11218Google Scholar
1973 Relationship between the inhibition constant (K1) and the concentration of inhibitor which causes 50 per cent inhibition (I50) of an enzymatic reactionBiochem Pharmacol 22 3099Google Scholar
2009 Mechanism of PTC124 activity in cell-based luciferase assays of nonsense codon suppressionProc Natl Acad Sci U S A 106 3585Google Scholar
2010 Molecular basis for the high-affinity binding and stabilization of firefly luciferase by PTC124Proc Natl Acad Sci U S A 107 4878Google Scholar
1989 A multisubstrate adduct inhibitor of a purine biosynthetic enzyme with a picomolar dissociation constantJ Med Chem 32 937Google Scholar
2005 Codon-optimized luciferase cDNA for mammalian gene expression in culture and in vivoMol Ther 11 435Google Scholar
2008 New approaches to in-cell detection of protein activity: genetically encoded chemiluminescence probes pave the way to robust HTS assaysACS Chem Biol 3 335Google Scholar
2008 High throughput screening (HTS) techniques: overview of applications in chemical biologyWiley Encylopedia of Chemical BiologyHoboken, N.JJohn Wiley & Sons, Inc
1995 Quantitative nonradioactive CAT assays using fluorescent BODIPY 1-deoxychloramphenicol substratesBiotechniques 19 488Google Scholar