A reporter gene assay for screening of PDE4 subtype selective inhibitors |
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Authors: | Bora Roop Singh Malik Renu Arya Ranjana Gupta Dikshi Singh Vibhuti Aggarwal Neeraj Dastidar Sunanda Ray Abhijit Saini Kulvinder Singh |
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Affiliation: | Department of Biotechnology, Ranbaxy Research Laboratories, Plot No. 20, Sector 18, Udyog Vihar Industrial Area, Gurgaon, Haryana 122001, India. roop.singh@ranbaxy.com |
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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. |
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Keywords: | Phosphodiesterase Stable cell lines Cellular localization Reporter gene assay |
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