title: A cell-based PDE4 assay in 1536-well plate format for high-throughput screening.
abstract: The cyclic nucleotide phosphodiesterases (PDEs) are intracellular enzymes that catalyze the hydrolysis of 3,'5'-cyclic nucleotides, such as cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), to their corresponding 5'nucleotide monophosphates. These enzymes play an important role in controlling cellular concentrations of cyclic nucleotides and thus regulate a variety of cellular signaling events. PDEs are emerging as drug targets for several diseases, including asthma, cardiovascular disease, attention-deficit hyperactivity disorder, Parkinson's disease, and Alzheimer's disease. Although biochemical assays with purified recombinant PDE enzymes and cAMP or cGMP substrate are commonly used for compound screening, cell-based assays would provide a better assessment of compound activity in a more physiological context. The authors report the development and validation of a new cell-based PDE4 assay using a constitutively active G-protein-coupled receptor as a driving force for cAMP production and a cyclic nucleotide-gated cation channel as a biosensor in 1536-well plates.
authors.affiliation: NIH Chemical Genomics Center, National Human Genome Research Institute, NIH, Bethesda, Maryland 20892-3370, USA.
authors.forename: Steven A
authors.initials: SA
authors.lastname: Titus
authors.forename: Xiao
authors.initials: X
authors.lastname: Li
authors.forename: Noel
authors.initials: N
authors.lastname: Southall
authors.forename: Jianming
authors.initials: J
authors.lastname: Lu
authors.forename: James
authors.initials: J
authors.lastname: Inglese
authors.forename: Michael
authors.initials: M
authors.lastname: Brasch
authors.forename: Christopher P
authors.initials: CP
authors.lastname: Austin
authors.forename: Wei
authors.initials: W
authors.lastname: Zheng
mesh_terms: D002412:Cations; D006152:Cyclic GMP / Q000737:chemistry; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4 / Q000378:metabolism*; D004353:Drug Evaluation, Preclinical / Q000295:instrumentation* / Q000379:methods*; D006801:Humans; D020128:Inhibitory Concentration 50; D007473:Ion Channels; D008954:Models, Biological; D058988:Phosphodiesterase 4 Inhibitors; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology*; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry; D015398:Signal Transduction; D013972:Thyrotropin / Q000378:metabolism; D013997:Time Factors; D014162:Transfection
chemical_list: D002412:Cations; D007473:Ion Channels; D058988:Phosphodiesterase 4 Inhibitors; D010726:Phosphodiesterase Inhibitors; D043562:Receptors, G-Protein-Coupled; D011994:Recombinant Proteins; D013972:Thyrotropin; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4; D006152:Cyclic GMP