Optimization and validation of a reporter gene assay for screening of phosphodiesterase inhibitors in a high throughput system.
Phosphodiesterases (PDEs) hydrolyze cyclic nucleotides, cyclic adenosine monophosphate (cAMP) and guanosine monophosphate (cGMP) into inactive 5' monophosphates, and exist as 11 families. Inhibitors of PDEs allow the elevation of cAMP and cGMP, which leads to a variety of cellular effects including airway smooth muscle relaxation and inhibition of cellular inflammation or of immune responses. PDE4 inhibitors specifically prevent the hydrolysis of cAMP. We have validated the manually developed reporter gene assay in a high-throughput screening format that allows for fast and cost-effective identification of potential inhibitors of PDE4 isozymes. The assay is sensitive and robust, with a Z' value of >0.5. The assay is also amenable to 384-well format.
Nanda
Kamna
K
Department of Pharmacology, Ranbaxy Research Laboratories, Gurgaon, Haryana, India. kamna.nanda@ranbaxy.com
Chatterjee
Mou
M
Arya
Ranjana
R
Mukherjee
Shohini
S
Saini
Kulvinder Singh
KS
Dastidar
Sunanda
S
Ray
Abhijit
A
D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism; D006152:Cyclic GMP / Q000378:metabolism; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4 / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008156:Luciferases / Q000235:genetics / Q000378:metabolism*; D058988:Phosphodiesterase 4 Inhibitors; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015203:Reproducibility of Results; D020889:Rolipram / Q000494:pharmacology*; D014162:Transfection
D058988:Phosphodiesterase 4 Inhibitors; D010726:Phosphodiesterase Inhibitors; D011993:Recombinant Fusion Proteins; D000242:Cyclic AMP; D008156:Luciferases; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4; C517360:PDE4B protein, human; D006152:Cyclic GMP; D020889:Rolipram