| Pharmacological Characterization of 5-Hydroxytryptamine2b Receptor in Evaluation of Aequorin for Miniaturized G-protein-coupled Receptor High Throughput Screening |
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Presentation: P03005 Session: Robotic Screening & Automation Technologies - Poster SessionMark A. Gilchrist II, Angela Cacace, George Hansen, and David G. Harden, Bristol-Myers Squibb Company Presenting Author: Mark Gilchrist II, Bristol-Myers Squibb Company - United States Fluorescent detection of calcium mobilization has been used successfully to identify modulators of GPCRs; however, inherent issues with fluorescence may limit its potential for HTS miniaturization. The data presented here demonstrate that the calcium-sensitive photoprotein aequorin, when compared to FLUO-4 in the same cellular background, allows for miniaturization of functional kinetic calcium flux assays, where rank order, potency, and efficacy were maintained for a series of diverse small molecule modulators. Low-volume 384- and 1536-well aequorin assays were implemented by integration of acoustic dispensing (Echo 550™) and kinetic flash luminometry (CyBi Lumax™). The high signal-to-background ratio of aequorin contributed to acceptable screening statistics in miniaturized format, and cell density modulation did not appear to alter pharmacology, which may be useful for improving sensitivity when screening orphan receptors. |