title: A reporter gene assay for screening of PDE4 subtype selective inhibitors.
abstract: Phosphodiesterase (PDE) constitutes a superfamily of enzymes that catalyze the hydrolysis of cAMP and cGMP into their corresponding monophosphates and play an important role in diverse physiological functions. The present study provides a process for identifying PDE4 subtypes selective inhibitors using a reporter gene assay. Stable recombinant HEK-293 cell lines expressing high levels of PDE4A4B, PDE4B2A, and PDE4D3 subtypes individually were generated. Transient transfection of pCRE-Luc plasmid, harboring luciferase reporter gene under the control of cAMP response element (CRE)-binding sequence, into these stable recombinant cell lines followed by treatment with PDE4 inhibitor, resulted in a dose dependent increase in luciferase activity. This methods provide a novel, simple and sensitive assay for high throughput screening of PDE4 subtype selective inhibitors for treatment of asthma and COPD.
authors.affiliation: Department of Biotechnology, Ranbaxy Research Laboratories, Plot No. 20, Sector 18, Udyog Vihar Industrial Area, Gurgaon, Haryana 122001, India. roop.singh@ranbaxy.com
authors.forename: Roop Singh
authors.initials: RS
authors.lastname: Bora
authors.forename: Renu
authors.initials: R
authors.lastname: Malik
authors.forename: Ranjana
authors.initials: R
authors.lastname: Arya
authors.forename: Dikshi
authors.initials: D
authors.lastname: Gupta
authors.forename: Vibhuti
authors.initials: V
authors.lastname: Singh
authors.forename: Neeraj
authors.initials: N
authors.lastname: Aggarwal
authors.forename: Sunanda
authors.initials: S
authors.lastname: Dastidar
authors.forename: Abhijit
authors.initials: A
authors.lastname: Ray
authors.forename: Kulvinder Singh
authors.initials: KS
authors.lastname: Saini
mesh_terms: D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D000631:Aminopyridines / Q000494:pharmacology; D001549:Benzamides / Q000494:pharmacology; D002460:Cell Line; D003001:Cloning, Molecular; D000242:Cyclic AMP / Q000378:metabolism; D017362:Cyclic AMP Response Element-Binding Protein / Q000235:genetics; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4; D003521:Cyclopropanes / Q000494:pharmacology; D003593:Cytoplasm / Q000201:enzymology; D004305:Dose-Response Relationship, Drug; D006801:Humans; D007150:Immunohistochemistry; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008156:Luciferases / Q000235:genetics / Q000378:metabolism*; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid; D020889:Rolipram / Q000494:pharmacology; D014162:Transfection; D020298:U937 Cells
chemical_list: D000631:Aminopyridines; D001549:Benzamides; D017362:Cyclic AMP Response Element-Binding Protein; D003521:Cyclopropanes; D007527:Isoenzymes; D010726:Phosphodiesterase Inhibitors; D011993:Recombinant Fusion Proteins; C424423:Roflumilast; D000242:Cyclic AMP; D008156:Luciferases; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4; D020889:Rolipram