© 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 1 Biotechnol. J. 2008, 3 DOI 10.1002/biot.200800102 www.biotechnology-journal.com 1 Introduction At the stage of lead generation for drug discovery, the combinatorial chemistry method and the high- throughput screening (HTS) method are being suc- cessfully utilized. Such methods are able to identi- fy several hit compounds from a huge chemical li- brary that usually consists of one hundred thou- sand to one million chemical compounds. Lead-like hits derived from HTS campaigns provide good starting points for lead optimization. Pharmaceuti- cal research relies on large-scale library screening for potential drug candidates. The scale and intri- cacy of selecting compounds out of these massive libraries require flexible, fast and reliable automa- tion. HTS techniques call for a reliable and accu- rate detection of output parameters. The reporter gene assay (RGA) is an assay that can be utilized for hit picking from a random library of com- pounds. The phosphodiesterases (PDEs) are a group of 11 families of metallophosphohydrolases that hydrolyze adenosine 3’5’-cyclic AMP (cAMP) and guanosine 3’5’-cyclic monophosphate (cGMP) to their inactive 5’monophosphates [1]. Inhibition of cyclic nucleotide PDEs causes elevation of cAMP/cGMP within cells. Therefore, inhibition of PDE is a useful way of producing a variety of cellu- lar effects and can influence inflammatory cell ac- tivation, immune cell activation and smooth muscle contractile responses. Each family of PDEs has varying selectivity for cAMP or cGMP,and is char- acterized by a unique combination of enzymatic characteristics and pharmacological inhibitory profiles.Amongst the PDEs, PDE4, PDE7 and PDE8 are specific for cAMP. The PDE4 family represents the largest PDE family, comprising four genes (PDE4A, PDE4B, Technical Report Optimization and validation of a reporter gene assay for screen- ing of phosphodiesterase inhibitors in a high throughput system Kamna Nanda 1 , Mou Chatterjee 1 , Ranjana Arya 2 , Shohini Mukherjee 2 , Kulvinder Singh Saini 2 , Sunanda Dastidar 1 and Abhijit Ray 1 1 Department of Pharmacology, Ranbaxy Research Laboratories, Gurgaon, Haryana, India 2 Department of Biotechnology, Ranbaxy Research Laboratories, Gurgaon, Haryana, India Keywords: High-throughput assay · Phosphodiesterase · Reporter gene assay 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 val- idated the manually developed reporter gene assay in a high-throughput screening format that al- lows 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. Correspondence: Ms. Kamna Nanda, Department of Pharmacology, NDDR, R &D-III, Ranbaxy Research Laboratories, Plot–20, Sector-18, Udhyog Vihar Industrial Area, Gurgaon, Haryana-122001, India E-mail: kamna.nanda@ranbaxy.com Fax: +91-124-2343544 Abbreviations: cAMP, cyclic adenosine monophosphate; cGMP, cyclic guanosine monophosphate; HTS, high throughput screening; PDE, phos- phodiesterase; RGA, reporter gene assay; RLU, relative luminescence units; S/B, signal to background ratio Received 24 April 2008 Revised 16 June 2008 Accepted 20 June 2008