A reporter gene assay for screening of PDE4 subtype selective inhibitors.
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.
Bora
Roop Singh
RS
Department of Biotechnology, Ranbaxy Research Laboratories, Plot No. 20, Sector 18, Udyog Vihar Industrial Area, Gurgaon, Haryana 122001, India. roop.singh@ranbaxy.com
Malik
Renu
R
Arya
Ranjana
R
Gupta
Dikshi
D
Singh
Vibhuti
V
Aggarwal
Neeraj
N
Dastidar
Sunanda
S
Ray
Abhijit
A
Saini
Kulvinder Singh
KS
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
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