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Optimization and validation of a reporter gene assay for screening of phosphodiesterase inhibitors in a high throughput system
Author(s) -
Nanda Kamna,
Chatterjee Mou,
Arya Ranjana,
Mukherjee Shohini,
Saini Kulvinder Singh,
Dastidar Sunanda,
Ray Abhijit
Publication year - 2008
Publication title -
biotechnology journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.144
H-Index - 84
eISSN - 1860-7314
pISSN - 1860-6768
DOI - 10.1002/biot.200800102
Subject(s) - phosphodiesterase , guanosine , high throughput screening , reporter gene , guanosine monophosphate , cyclic nucleotide phosphodiesterase , cyclic guanosine monophosphate , pde10a , adenosine , isozyme , biochemistry , chemistry , cyclic nucleotide , biology , nucleotide , enzyme , gene , gene expression , endocrinology , nitric oxide
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.