Source:http://linkedlifedata.com/resource/pubmed/id/20161830
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| rdf:type | |
| lifeskim:mentions | |
| pubmed:dateCreated |
2010-2-17
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| pubmed:abstractText |
Cell-based functional assays used for compound screening and lead optimization play an important role in drug discovery for G-protein coupled receptors (GPCRs). Cell-based assays can define the role of a compound as an agonist, antagonist or inverse agonist and can provide detailed information about the potency and efficacy of a compound. In addition, cell-based screens can be used to identify allosteric modulators that interact with sites other than the binding site of the endogenous ligand. Intracellular calcium assays which use a fluorescent calcium binding dye (such as Fluo-3, Fluo-4 or Fura-2) have been used in compound screening campaigns to measure the activity of Gq-coupled GPCRs. However, such screening methodologies require a special instrumentation to record the rapid change in intracellular free calcium concentration over time. The radioactive inositol 1,4,5- triphosphate (IP(3)) assay measures (3)H-inositol incorporation and is another traditional assay for the assessment of Gq-coupled GPCR activity, but it is not suitable for screening of large size compound collections because it requires a cell wash step and generates radioactive waste. To avoid these limitations, we have optimized and miniaturized a TR-FRET based IP-One assay that measures inositol monophosphate in a 1536-well plate format. This assay is homogenous, non-radioactive and does not require a kinetic readout. It has been tested with the cell lines expressing M(1) acetylcholine, FFAR1, vasopressin V1b, or Neuropeptide S receptors. The activities of antagonists determined in the IP-One assay correlated well with these measured in the intracellular calcium assay while the correlation of agonist activities might vary from cell line to cell line. This IP-One assay offers an alternative method for high throughput screening of Gq-coupled GPCRs without using costly kinetic plate readers.
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| pubmed:grant | |
| pubmed:commentsCorrections |
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| pubmed:language |
eng
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| pubmed:journal | |
| pubmed:status |
PubMed-not-MEDLINE
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| pubmed:issn |
1875-3973
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| pubmed:author | |
| pubmed:issnType |
Electronic
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| pubmed:volume |
1
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| pubmed:owner |
NLM
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| pubmed:authorsComplete |
Y
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| pubmed:pagination |
70-8
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| pubmed:dateRevised |
2010-9-28
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| pubmed:year |
2008
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| pubmed:articleTitle |
Comparison on functional assays for Gq-coupled GPCRs by measuring inositol monophospate-1 and intracellular calcium in 1536-well plate format.
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| pubmed:affiliation |
NIH Chemical Genomics Center, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD 20892-3370, USA.
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| pubmed:publicationType |
Journal Article
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