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An Offline-Addition Format for Identifying GPCR Modulators by Screening 384-Well Mixed Compounds in the FLIPR

Advanced Technology, Global Pharmaceutical Research and Development, Abbott Laboratories, Abbott Park, IL

Betsy Mammen

Advanced Technology, Global Pharmaceutical Research and Development, Abbott Laboratories, Abbott Park, IL

Martin Schmidt

CNS Discovery Research, Abbott GmbH & Co. KG, Ludwigshafen, Germany

Bernd Otterstaetter

CNS Discovery Research, Abbott GmbH & Co. KG, Ludwigshafen, Germany

Willi Amberg

CNS Discovery Research, Abbott GmbH & Co. KG, Ludwigshafen, Germany

Wolfgang Wernet

CNS Discovery Research, Abbott GmbH & Co. KG, Ludwigshafen, Germany

James L. Kofron

Advanced Technology, Global Pharmaceutical Research and Development, Abbott Laboratories, Abbott Park, IL

David J. Burns

Advanced Technology, Global Pharmaceutical Research and Development, Abbott Laboratories, Abbott Park, IL

Usha Warrior

Advanced Technology, Global Pharmaceutical Research and Development, Abbott Laboratories, Abbott Park, IL

Although fluorescence imaging plate reader (FLIPR)-based assays have been widely used in high-throughput screening, improved efficiencies in throughput and fidelity continue to be investigated. This study presents an offline compound addition protocol coupled with a testing strategy using mixtures of compounds in a 384-well format to identify antagonists of the neurokinin-1 receptor expressed in the human astrocytoma cell line (U373 MG). Substance P evoked a concentration-dependent increase in intracellular cellular Ca²⁺ with an EC₅₀ value of 0.30 ± 0.17 nM, which was inhibited by neurokinin-1 (NK1) antagonists L-733,060 and L-703,606. Test compounds, as mixtures of 10 compounds/well, were added to the cells offline using an automated dispensing unit and incubated prior to performing the assay in the FLIPR. Using the offline protocol, a higher through put of ~200,000 compounds was achieved in an 8-h working day, and several novel structural classes of compounds were identified as antagonists for the NK1 receptor. These studies demonstrate that the offline compound addition format using a mixture of compounds in a 384-well FLIPR assay provides an efficient platform for screening and identifying modulators for G-protein-coupled receptors. (Journal of Biomolecular Screening 2005:46-55)

Key Words: G-protein-coupled receptor • FLIPR • intracellular calcium release • neurokinin • high-throughput screening

Journal of Biomolecular Screening, Vol. 10, No. 1, 46-55 (2005)
DOI: 10.1177/1087057104270017


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