Miniaturization of Whole Live Cell-Based GPCR Assays Using Microdispensing and Detection Systems

Kornienko, Oleg; Lacson, Raul; Kunapuli, Priya; Schneeweis, Jonathan; Hoffman, Ira; Smith, Todd; Alberts, Melissa; Inglese, James; Strulovici, Berta

doi:10.1177/1087057103260070

Cited by 35 publications

(30 citation statements)

References 23 publications

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“…The second messengers most commonly measured are cAMP, IP3, and Ca 2ϩ . [157][158][159][160] Detecting these molecules measures functional responses of GPCRs coupled to the most predominant G proteins including G␣ s , G␣ i , G␣ o , and G␣ q . Using these assays, one can identify full agonists, partial agonists, and inverse agonists at both native and recombinant receptors.…”

Section: Cell-based Assays Measuring Second Messengers and Reporter Gmentioning

confidence: 99%

Emerging Concepts of Guanine Nucleotide-Binding Protein-Coupled Receptor (GPCR) Function and Implications for High Throughput Screening

Eglen

Bossé

Reisine³

2007

ASSAY and Drug Development Technologies

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Guanine nucleotide binding protein (G protein) coupled receptors (GPCRs) comprise one of the largest families of proteins in the human genome and are a target for 40% of all approved drugs. GPCRs have unique structural motifs that allow them to interact with a wide and diverse series of extracellular ligands, as well as intracellular proteins, G proteins, receptor activity-modifying proteins, arrestins, and indeed other receptors. This distinctive structure has led to numerous efforts to discover drugs against GPCRs with targeted therapeutic uses. Such "designer" drugs currently include allosteric regulators, inverse agonists, and drugs targeting hetero-oligomeric complexes. Moreover, the large family of orphan GPCRs provides a rich and novel field of targets to discover drugs with unique therapeutic properties. The numerous technologies to discover GPCR drugs have also greatly advanced over the years, facilitating compound screening against known and orphan GPCRs, as well as in the identification of unique designer GPCR drugs. Indeed, high throughput screening (HTS) technologies employing functional cell-based approaches are now widely used. These include measurement of second messenger accumulation such as cyclic AMP, calcium ions, and inositol phosphates, as well as mitogen-activated protein kinase activation, protein-protein interactions, and GPCR oligomerization. This review focuses on how the improved understanding of the molecular pharmacology of GPCRs, coupled with a plethora of novel HTS technologies, is leading to the discovery and development of an entirely new generation of GPCR-based therapeutics.

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Section: Cell-based Assays Measuring Second Messengers and Reporter Gmentioning

confidence: 99%

Emerging Concepts of Guanine Nucleotide-Binding Protein-Coupled Receptor (GPCR) Function and Implications for High Throughput Screening

Eglen

Bossé

Reisine³

2007

ASSAY and Drug Development Technologies

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“…Ultra-high-throughput screening was performed in a 3456-well plate format 14 with a Discovery Island robot (Aurora Discovery, San Diego, CA). CHO/DAO cells were suspended in the assay buffer at 2.5 × 10 6 cell/mL and supplied for screening every 2 h (twice a day).…”

Section: Cell Line Establishment and Cell Culturementioning

confidence: 99%

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

et al. 2006

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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. (Journal of Biomolecular Screening 2006:481-487)

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“…The ranges of volumes above and below this criterion are those routinely encountered in constructing miniaturized assay samples. The instruments developed by Aurora Discovery for dispensing in these ranges of 0.5-100 nl and 100 nl to microliter have been found to be useful for a variety of assays for which miniaturization has been exploited to yield benefits including library screening for enzyme inhibition, 25 G protein-coupled receptor, 15 and nuclear transcription factor activities. 26 The goal of Aurora Discovery is to extend the applicability of HTS technologies to new areas of biological inquiry that would benefit from miniaturization.…”

Section: Discussionmentioning

confidence: 99%

“…Utility of these plates in fluorescence assays has been demonstrated for compound screening, 2 ADME toxicity assays, 14 and chemical library storage. 12 As benefits of COC for fluorescence become more widely known, its is hoped that they will be applied more broadly for miniaturized assay development, 15 lead optimization, 2 polymerase chain reaction, 16 single nucleotide polymoprhism detection, and other fluorescence-based assays performed in low-volume formats.…”

Section: A Key Miniaturization Technology: the High-density Multiwellmentioning

confidence: 99%

Piezo- and Solenoid Valve-Based Liquid Dispensing for Miniaturized Assays

Niles¹,

Coassin²

2005

ASSAY and Drug Development Technologies

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Miniaturization of biological assays requires dispensing liquids in the submicroliter range of volumes. Accuracy and reproducibility of dispensing this range depend on both the dispenser and the receptacle in which the assay is constructed. Miniaturization technologies developed by Aurora Discovery, Inc. (San Diego, CA) include high-density multiwell plates for assay samples and reagent storage, as well as piezo-based and solenoid valve-based liquid dispensers. Some basic principles of small-volume dispensing by jetting are described to provide context for dispenser design and function. Performance of the latest instruments incorporating these dispensing devices is presented.

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Miniaturization of Whole Live Cell-Based GPCR Assays Using Microdispensing and Detection Systems

Cited by 35 publications

References 23 publications

Emerging Concepts of Guanine Nucleotide-Binding Protein-Coupled Receptor (GPCR) Function and Implications for High Throughput Screening

Emerging Concepts of Guanine Nucleotide-Binding Protein-Coupled Receptor (GPCR) Function and Implications for High Throughput Screening

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

Piezo- and Solenoid Valve-Based Liquid Dispensing for Miniaturized Assays

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