Enzyme Fragment Complementation
DiscoveRx (www.discoverx. com) uses an enzyme fragment complementation strategy for high throughput cell-based GPCR screening. The technology relies on dividing the enzyme, -galactosidase (-gal), into two inactive fragments, ED and EA.
One of these (the alpha fragment, termed ED) is chemically conjugated to a second messenger. When both -gal fragments encounter each other they combine to form active enzyme, which hydrolyzes a substrate and emits a chemiluminescent or fluorescent signal.
"One of the major advantages of this type of assay is that it is nonradioactive and homogeneous," Richard M. Eglen, Ph.D., CSO, notes. "So, washing and separation steps are not required. The reagents are simply added and the signal read on common plate readers. This allows using automated fluid-handling systems for primary screening of ligands that modulate GPCR activation."
An added benefit of chemiluminescent signals, in particular, is that they can provide a large signal to background ratio, according to Dr. Eglen.
"The assay format is easily adapted to different fluid volumes needed in assays that are miniaturized for high density microtiter plates. Furthermore, the interference potential of compounds in screening libraries is very low, resulting in few false positives and high confirmed hit rates."
Dr. Eglen suggests that the technology also allows GPCR secondary messenger analyses. "In this case, we measure changes in intracellular cyclic AMP for Gs and Gi coupled GPCR receptors. The Gi coupled receptors are often quite difficult to assay, and an assay of this type provides a marked advantage.
"Using a different format, based on fluorescent polarization, DiscoveRx has also developed assays for IP3, a second messenger generated by Gq coupled GPCRs. Together, these assay formats are valuable for screening for novel ligands interacting at known and orphan receptors, particularly as one often needs to measure changes in constitutive activity of these receptors."