A cGMP-dependent protein kinase assay for high throughput screening based on time-resolved fluorescence resonance energy transfer

Activation of cyclic GMP-dependent protein kinase (cGK) is an important eve nt in the regulation of blood pressure and platelet function. Upstream sign als are the generation of nitric oxide (NO) by NO synthases and the subsequ ent rise in cyclic GMP levels mediated by NO-dependent guanylyl cyclases (G Cs). The identification of new cGK activators by high throughput screening (HTS) may lead to the development of a novel class of therapeutics for the treatment of cardiovascular diseases. Therefore, a homogeneous, nonradioact ive assay for cGK activity was developed using a biotinylated peptide deriv ed from vasodilator-stimulated phosphoprotein (VASP), a well-characterized natural cGK substrate. The phosphorylated peptide could be detected by a VA SP-specific monoclonal phosphoserine antibody and a fluorescent detection s ystem consisting of a europium-labeled secondary antibody and allophycocyan in (APC)-labeled streptavidin. Fluorescence resonance energy transfer (FRET ) from europium to APC was detected in a time-resolved fashion (TR-FRET). A ctivation and inhibition constants for known substances determined by this new fluorescence-based assay correlated well with published results obtaine d by conventional radioactive cGK activity assays. The assay proved to be s ensitive, robust, highly specific for cGK, and suitable for HTS in 96- and 384-well formats. This assay is applicable to purified enzymes as well as t o complex samples such as human platelet extracts.