G-Protein Coupled Receptors (GPCRs) represent large, diverse gene families found in fungi, plants and animals. GPCRs are involved in many critical cellular signaling processes in both normal and disease states. Since the receptor spans the cell membrane and the ligand binding site is exposed to blood flow in animals, GPCRs represent one of the most druggable drug target families for small molecule drug discovery. GPCR cellular signaling is initiated by second messengers: either through the production of adenosine 3’, 5’-cyclic monophosphate (cAMP), its reduction or production of inositol-1,4,5-triphosphate (IP3).
In today’s drug discovery programs, cAMP quantification is typically performed by homogeneous proximity assays that rely on the lytic release of cAMP generated by cellular signaling into an assay buffer where it competes with a labeled standard of cAMP for a limited number of antibody binding sites. AlphaScreen®, LANCE® and HTRF® are all examples of these types of assays. However, these methods are considered end-point assays in that they require cell lysis prior to detection of intracellular cAMP levels. The ability to study kinetic cellular function in live cells remains an attractive alternative for many scientists.
Luminescent reporter genes have been shown to provide increased sensitivity with a wide dynamic range and are easily adaptable to a high density microplate format amenable to automation. One such example is a recently developed biosensor based on firefly luciferase. The biomolecule exhibits high sensitivity in addition to ligand specificity allowing live-cell, real-time monitoring of cAMP kinetics. The ability to either stably or transiently transform the biosensor into a preferred cell line allows investigation of both endogenous and over-expressed GPCR targets. Technologies such as this should provide a means to gain critical insight into molecular processes from live-cell assays.
By, BioTek Instruments