title: Ultra-high-throughput screening for antagonists of a Gi-coupled receptor in a 2.2-microl 3,456-well plate format cyclicAMP assay.
abstract: 3',5'-Cyclic adenosine monophosphate (cAMP) is a common intracellular second messenger that enables cells to respond to external stimuli. Measurement of intracellular cAMP concentrations is thus widely used for studying guanosine triphosphate binding protein-coupled receptors (GPCRs), which make up a large class of pharmaceutical drug targets. Although several assay technologies exist to measure cAMP, most are not suitable for ultra-high-throughput screening (uHTS), as is often required for screening large (greater than 1 million) chemical libraries for the identification of suitable leads for drug development. Here we report that the enzyme fragment complementation assay, a homogeneous gain of signal assay based on complementation of two fragments of a beta-galactosidase enzyme, is compatible with uHTS requirements of a 2.2-microl total assay volume in 3,456-well plate format. We describe the miniaturization of this assay into 3,456-well plate format exhibiting comparable sensitivity and plate statistics to those of a 384-well assay and the application of this assay in uHTS for the identification of antagonists of a Gi-coupled receptor.
authors.affiliation: Department of Automated Biotechnology, Merck Research Laboratories, North Wales, PA 19454, USA. michael_weber@merck.com
authors.forename: Michael
authors.initials: M
authors.lastname: Weber
authors.forename: Lalitha
authors.initials: L
authors.lastname: Muthusubramaniam
authors.forename: Justin
authors.initials: J
authors.lastname: Murray
authors.forename: Ed
authors.initials: E
authors.lastname: Hudak
authors.forename: Oleg
authors.initials: O
authors.lastname: Kornienko
authors.forename: Eric N
authors.initials: EN
authors.lastname: Johnson
authors.forename: Berta
authors.initials: B
authors.lastname: Strulovici
authors.forename: Priya
authors.initials: P
authors.lastname: Kunapuli
mesh_terms: D001681:Biological Assay / Q000379:methods*; D019204:GTP-Binding Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry*; D008832:Microchemistry / Q000379:methods*; D012371:Robotics / Q000379:methods*; D013050:Spectrometry, Fluorescence / Q000379:methods*
chemical_list: D019204:GTP-Binding Proteins