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1.  A Multiplex Calcium Assay for Identification of GPCR Agonists and Antagonists 
Abstract
Activation of Gq protein-coupled receptors can be monitored by measuring the increase in intracellular calcium with fluorescent dyes. Recent advances in fluorescent kinetic plate readers and liquid-handling technology have made it possible to follow these transient changes in intracellular calcium in a 1,536-well plate format for high-throughput screening (HTS). Here, we have applied the latest generation of fluorescence kinetic plate readers to multiplex the agonist and antagonist screens of a G protein-coupled receptor (GPCR). This multiplexed assay format provides an efficient and cost-effective method for HTS of Gq-coupled GPCR targets.
doi:10.1089/adt.2009.0245
PMCID: PMC2893246  PMID: 20230302
2.  A Multiplex Calcium Assay for Identification of GPCR Agonists and Antagonists 
Activation of Gq protein-coupled receptors can be monitored by measuring the increase in intracellular calcium with fluorescent dyes. Recent advances in fluorescent kinetic plate readers and liquid-handling technology have made it possible to follow these transient changes in intracellular calcium in a 1536-well plate format for high-throughput screening. Here, we have applied the latest generation of fluorescence kinetic plate readers to multiplex the agonist and antagonist screens of a GPCR. This multiplexed assay format provides an efficient and cost-effective method for high-throughput screening of Gq-coupled GPCR targets.
doi:10.1089/adt.2009.0245
PMCID: PMC2893246  PMID: 20230302
GPCR; calcium assay; FDSS; multiplex; high-throughput screening
3.  A Dual-Fluorescence High-Throughput Cell Line System for Probing Multidrug Resistance 
Abstract
The efflux pump P-glycoprotein (ATP-binding cassette B1, multidrug resistance [MDR] 1, P-gp) has long been known to contribute to MDR against cancer chemotherapeutics. We describe the development of a dual-fluorescent cell line system to allow multiplexing of drug-sensitive and P-gp-mediated MDR cell lines. The parental OVCAR-8 human ovarian carcinoma cell line and the isogenic MDR NCI/ADR-RES subline, which stably expresses high levels of endogenous P-gp, were transfected to express the fluorescent proteins Discosoma sp. red fluorescent protein DsRed2 and enhanced green fluorescent protein, respectively. Co-culture conditions were defined, and fluorescent barcoding of each cell line allowed for the direct, simultaneous comparison of resistance to cytotoxic compounds in sensitive and MDR cell lines. We show that this assay system retains the phenotypes of the original lines and is suitable for multiplexing using confocal microscopy, flow cytometry, or laser scanning microplate cytometry in 1,536-well plates, enabling the high-throughput screening of large chemical libraries.
doi:10.1089/adt.2008.165
PMCID: PMC2814070  PMID: 19548831
4.  Identification of Pregnane X Receptor Ligands Using Time-Resolved Fluorescence Resonance Energy Transfer and Quantitative High-Throughput Screening 
Abstract
The human pregnane X nuclear receptor (PXR) is a xenobiotic-regulated receptor that is activated by a range of diverse chemicals, including antibiotics, antifungals, glucocorticoids, and herbal extracts. PXR has been characterized as an important receptor in the metabolism of xenobiotics due to induction of cytochrome P450 isozymes and activation by a large number of prescribed medications. Developing methodologies that can efficiently detect PXR ligands will be clinically beneficial to avoid potential drug–drug interactions. To facilitate the identification of PXR ligands, a time-resolved fluorescence resonance energy transfer (TR-FRET) assay was miniaturized to a 1,536-well microtiter plate format to employ quantitative high-throughput screening (qHTS). The optimized 1,536-well TR-FRET assay showed Z′-factors of ≥0.5. Seven- to 15-point concentration–response curves (CRCs) were generated for 8,280 compounds using both terbium and fluorescein emission data, resulting in the generation of 241,664 data points. The qHTS method allowed us to retrospectively examine single concentration screening datasets to assess the sensitivity and selectivity of the PXR assay at different compound screening concentrations. Furthermore, nonspecific assay artifacts such as concentration-based quenching of the terbium signal and compound fluorescence were identified through the examination of CRCs for specific emission channels. The CRC information was also used to define chemotypes associated with PXR ligands. This study demonstrates the feasibility of profiling thousands of compounds against PXR using the TR-FRET assay in a high-throughput format.
doi:10.1089/adt.2009.193
PMCID: PMC3116688  PMID: 19505231
5.  A dual-fluorescence high-throughput cell line system for probing multidrug resistance 
The efflux pump P-glycoprotein (ABCB1, MDR1, P-gp) has long been known to contribute to multidrug resistance (MDR) against cancer chemotherapeutics. We describe the development of a dual-fluorescent cell line system to allow multiplexing of drug-sensitive and P-gp-mediated MDR cell lines. The parental OVCAR-8 human ovarian carcinoma cell line and the isogenic MDR NCI/ADR-RES subline, which stably expresses high levels of endogenous P-gp, were transfected to express the fluorescent proteins DsRed2 and EGFP respectively. Co-culture conditions were defined and fluorescent barcoding of each cell line allowed for the direct, simultaneous comparison of resistance to cytotoxic compounds in sensitive and MDR cell lines. We show that this assay system retains the phenotypes of the original lines and is suitable for multiplexing using confocal microscopy, flow cytometry or laser-scanning microplate cytometry in 1,536-well plates, enabling the high-throughput screening (HTS) of large chemical libraries.
doi:10.1089/adt.2008.165
PMCID: PMC2814070  PMID: 19548831
6.  A Robotic Platform for Quantitative High-Throughput Screening 
Abstract
High-throughput screening (HTS) is increasingly being adopted in academic institutions, where the decoupling of screening and drug development has led to unique challenges, as well as novel uses of instrumentation, assay formulations, and software tools. Advances in technology have made automated unattended screening in the 1,536-well plate format broadly accessible and have further facilitated the exploration of new technologies and approaches to screening. A case in point is our recently developed quantitative HTS (qHTS) paradigm, which tests each library compound at multiple concentrations to construct concentration-response curves (CRCs) generating a comprehensive data set for each assay. The practical implementation of qHTS for cell-based and biochemical assays across libraries of > 100,000 compounds (e.g., between 700,000 and 2,000,000 sample wells tested) requires maximal efficiency and miniaturization and the ability to easily accommodate many different assay formats and screening protocols. Here, we describe the design and utilization of a fully integrated and automated screening system for qHTS at the National Institutes of Health's Chemical Genomics Center. We report system productivity, reliability, and flexibility, as well as modifications made to increase throughput, add additional capabilities, and address limitations. The combination of this system and qHTS has led to the generation of over 6 million CRCs from > 120 assays in the last 3 years and is a technology that can be widely implemented to increase efficiency of screening and lead generation.
doi:10.1089/adt.2008.150
PMCID: PMC2651822  PMID: 19035846
7.  A 1,536-Well-Based Kinetic HTS Assay for Inhibitors of Schistosoma mansoni Thioredoxin Glutathione Reductase 
Abstract
Schistosomiasis is a major neglected tropical disease that currently affects over 200 million people and leads to over 200,000 annual deaths. Schistosoma mansoni parasites survive in humans in part because of a set of antioxidant enzymes that continuously degrade reactive oxygen species produced by the host. A principal component of this defense system has been recently identified as thioredoxin glutathione reductase (TGR), a parasite-specific enzyme that combines the functions of two human counterparts, glutathione reductase and thioredoxin reductase, and as such this enzyme presents an attractive new target for anti-schistosomiasis drug development. Herein, we present the development of a highly miniaturized and robust screening assay for TGR. The 5-μl final volume assay is based on the Ellman reagent [5,5′-dithiobis(2-nitrobenzoic acid) (DTNB)] and utilizes a high-speed absorbance kinetic read to minimize the effect of dust, absorbance interference, and meniscus variation. This assay is further applicable to the testing of other redox enzymes that utilize DTNB as a model substrate.
doi:10.1089/adt.2008.149
PMCID: PMC2669305  PMID: 18665782
8.  A 1536-well Based Kinetic HTS Assay for Inhibitors of Schistosoma mansoni Thioredoxin Glutathione Reductase 
Schistosomiasis is a major neglected tropical disease that currently affects over 200 million people and leads to over 200,000 annual deaths. Schistosoma mansoni parasites survive in humans due in part to a set of antioxidant enzymes that continuously degrade reactive oxygen species produced by the host. A principle component of this defense system has been recently identified as thioredoxin glutathione reductase (TGR), a parasite-specific enzyme that combines the functions of two human counterparts, gluotathione reductase and thioredoxin reductase, and as such this enzyme presents an attractive new target for anti-schistosomiasis drug development. Herein, we present the development of highly-miniaturized and robust screening assay for TGR. The 5 μL final volume assay is based on the Ellman’s reagent (5,5′ dithiobis(2-nitrobenzoic acid), DTNB) and utilizes a high-speed absorbance kinetic read to minimize the effect of dust, absorbance interference, and meniscus variation. This assay is further applicable to the testing of other redox enzymes which utilize DTNB as a model substrate.
doi:10.1089/adt.2008.149
PMCID: PMC2669305  PMID: 18665782
9.  A Robotic Platform for Quantitative High Throughput Screening 
High-throughput screening (HTS) is increasingly being adopted in academic institutions, where the decoupling of screening and drug development has led to unique challenges, as well as novel uses of instrumentation, assay formulations, and software tools. Advances in technology have made automated unattended screening in the 1536-well plate format broadly accessible and have further facilitated the exploration of new technologies and approaches to screening. A case in point is our recently-developed quantitative high-throughput screening (qHTS) paradigm which tests each library compound at multiple concentrations to construct concentration-response curves (CRCs) generating a comprehensive data set for each assay (Inglese et al, Proc. Natl. Acad. Sci USA 103, 11473–11478). The practical implementation of qHTS for cell-based and biochemical assays across libraries of >100,000 compounds (e.g. between 700,000 –2,000,000 sample wells tested) requires maximal efficiency and miniaturization, and the ability to easily accommodate many different assay formats and screening protocols. Here, we describe the design and utilization of a fully-integrated and automated screening system for qHTS at the NIH Chemical Genomics Center. We report system productivity, reliability, and flexibility, as well as modifications made to increase throughput, add additional capabilities, and address limitations. The combination of this system and qHTS has lead to the generation of over 6 million CRCs from >120 assays in the last three years, and is a technology that can be widely implemented to increase efficiency of screening and lead generation.
doi:10.1089/adt.2008.150
PMCID: PMC2651822  PMID: 19035846
quantitative high-throughput screening; screening; qHTS; 1536-well plate; laboratory automation; interleaved screens; laser cytometer

Results 1-9 (9)