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A Novel Homogeneous, Fluorescence-based Technology for Dual Measure of Phosphodiesterases and Their Downstream Effector Kinase Activities in Biochemical and Cell-based AssaysTélécharger
January 26, 2010
Authors: Frauke Rininsland, Wendy Weatherford, Gyrasol Technologies, Santa Fe, NM, USA; Peter Banks, BioTek Instruments, Winooski, VT, USA
Measure of post translational modifications, such as the addition of phosphates, their removal or the cleavage of biological substrates provides the most meaningful assessment of a given signal transduction status of a cell. Major transducers of cellular signaling processes are the second messengers cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) via their interaction with downstream targets such as Protein Kinase A (PKA) and Protein Kinase G (PKG). The balance of cAMP and cGMP is tightly controlled by phosphodiesterases (PDEs) through their hydrolysis of the cyclic nucleotide bonds. Here we present a fluorescent sensing approach with which PDE as well as kinase activities can be monitored with one platform. The platform is based on photo-induced electron transfer quench of fluor labeled substrates by a metal ion upon its association to a phosphoryl group present on the substrate. Since the mechanism of electron transfer does not require spectral overlap between donor and acceptor molecules, fluorescence of any fluor can be quenched by one sensor. Hence the platform is ideally suited for multiplexed measurement of various substrate modifications. We show highly sensitive and simultaneous detection of cAMP and cGMP hydrolyses by PDE1C in biochemical assays as well as in lysates of rat brain. Assays are homogeneous and when measured in endpoint mode deliver Z’ factors of 0.8. In addition to being suitable for robotics, the platform can be run in kinetic mode, thus greatly simplifying mode of action analysis of inhibitors. Monitoring of PDE4 and PDE5 activities within one well in the presence of specific inhibitors produced IC50 values, which closely match reference values. Lastly, we demonstrate the correlation between PKA-mediated phosphorylation of fluor-labeled Kemptide with cAMP hydrolysis within lysates of rat brain in one experiment. In conclusion, the platform promises to be a cost effective new tool to gain a better understanding of a drug’s action on catalytic events within interconnected cellular pathways.