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HitHunter IP3 assay for GPCR screening using the PHERAstar FS

Lindy Kauffman, Sherrylyn De La Llera DiscoverX Corporation 05/2006

The Hithunter® IP3 assay is a robust, sensitive and specific tool for measuring cellular D-myo-inositol 1,4,5-triphosphate. The signal is based on competitive binding between an IP3 fluorescent tracer and an unlabeled IP3 from cell lysate or IP3 standard. The signal is read as a change in fluorescence polarization and is inversely proportional to the amount of IP3 in cell lysates. The standard curve signal to background is >5, and the EC50 is 13 nM. The signal can be measured immediately or up to 16 hours later.


Conclusion

 

GPCRs are critical targets in HTS drug discovery and GPCR signaling can be examined by direct quantitation of IP3 by applying the HitHunter® IP3 kit. Good results were obtained on the PHERAstar FS multimode microplate reader which is designed to read all leading HTS detection modes in formats up to 1536. The high degree of sensitivity, easy-to-use software, robust hardware and optimized detection systems make the PHERAstar FS ideal for GPCR analyses in the high-throughput assay environment.

 

The PHERAstar was run in FP mode for the HitHunter® IP3 assay, which uses FP for sensitivite detection and ease of use. The assay is ideal for the detection of basal levels of IP3 in cell lysates, as well as low levels for IP3 induction. Once the cells are lysed, the stable assay signal can be read the same day or overnight. There are few robust IP3 assays currently available and none easily scalable to 1536 automation. DiscoverX offers this assay to give maximum precision and reliability for GPCR screening. 


For more information on DiscoverX assays please refer to the website: www.discoverx.com


HitHunter, DiscoverX and the DiscoverX logo are registered trademarks of the DiscoverX Corporation.

G-protein coupled receptor (GPCR) activation regulates cell signaling via several second messengers, including cAMP, inositol phospholipids and calcium. Upon activation of a Gq-type receptor, inositol-triphosphate (IP3) is formed that is used by the DiscoverX HitHunter assay to identify Gq-acting drugs.

 

The fluorescence polarization assay uses an IP3-binding protein saturated with fluorophore-coupled IP3. The protein-IP3-fluorophore complex exhibits low depolarization of fluorescence emission light due to its high molecular weight and small rotational movement. However, in the presence of native IP3, the fluorescent IP3 tracer competes with the native molecule and is released from the IP3-binding protein. The free tracer has a much lower molecular weight and, therefore, a higher rotational movement which finally results in depolarization of emission light.

 

A standard curve of IP3 was measured in 384 well format to validate the assay on the PHERAstar® microplate reader. With increasing analyte concentration, the fluorescence polarization value decreased and an EC50 of 13 nM was determined.

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