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Development of a sensitive enzyme fragment complementation assay for cyclic adenosine 3', 5' monophosphate and its validation for pharmacological screening at G protein-coupled receptors

Key words: cAMP • GPCR enzyme fragment complementation LEADseeker assay miniaturization


G protein-coupled receptors (GPCRs) are the largest family of transmembrane receptors and are responsible for the transduction of a diverse range of extracellular signals. Up to 40% of clinically marketed drugs are active at this receptor family; however, these drugs exhibit their activity at less than 10% of known GPCRs. Thus, sensitive high-throughput GPCR screening assays are required to associate the many orphan GPCRs with disease to potentially identify novel pharmaceutical agents.

Here, we describe an assay for screening at GPCRs based on enzyme fragment complementation (EFC) technology and cyclic adenosine 3’, 5’ monophosphate (cAMP) measurement. The data show the cAMP II HitHunter Chemiluminescence Assay has a sensitivity of ~ 1 nM when carried out in 1536-well microplates and imaged using the LEADseeker™ Multimodality Imaging System.


Materials
Products used

LEADseeker Multimodality Imaging System 18-1140-71

cAMP II HitHunter Chemiluminescence 90-0032-03
Assay, 1536 wells


Other materials required
Krebs-Henseleit buffer

Phosphate buffered saline (PBS) solution

Dulbecco’s Modified Eagle’s Medium

DMSO

Ethanol

Greiner Bio-one Lumitrac™ 200 microplates, 1536-wells

Biomek™ FX Laboratory Automated Workstation (Beckman Coulter)

CyBi™-well (Cy-Bio)

Chinese hamster ovary (CHO-K1)
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