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 event in the regulation of blood pressure and platelet function. Upstream signals are the generation of nitric oxide (NO) by NO synthases and the subsequent rise in cyclic GMP levels mediated by NO-dependent guanylyl cyclases (GCs). 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, nonradioactive assay for cGK activity was developed using a biotinylated peptide derived from vasodilator-stimulated phosphoprotein (VASP), a well-characterized natural cGK substrate. The phosphorylated peptide could be detected by a VASP-specific monoclonal phosphoserine antibody and a fluorescent detection system consisting of a europium-labeled secondary antibody and allophycocyanin (APC)-labeled streptavidin. Fluorescence resonance energy transfer (FRET) from europium to APC was detected in a time-resolved fashion (TR-FRET). Activation and inhibition constants for known substances determined by this new fluorescence-based assay correlated well with published results obtained by conventional radioactive cGK activity assays. The assay proved to be sensitive, 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 to complex samples such as human platelet extracts.
Bader
B
B
Vasopharm BIOTECH GmbH, Wuerzburg, Germany.
Butt
E
E
Palmetshofer
A
A
Walter
U
U
Jarchau
T
T
Drueckes
P
P
D000470:Alkaloids / Q000494:pharmacology; D000595:Amino Acid Sequence; D001331:Automation; D001792:Blood Platelets / Q000201:enzymology / Q000378:metabolism; D015153:Blotting, Western; D002227:Carbazoles; D015815:Cell Adhesion Molecules / Q000378:metabolism; D006152:Cyclic GMP / Q000378:metabolism; D017869:Cyclic GMP-Dependent Protein Kinases / Q000378:metabolism*; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D004789:Enzyme Activation; D006801:Humans; D007211:Indoles; D019169:Jurkat Cells; D008840:Microfilament Proteins; D008954:Models, Biological; D008969:Molecular Sequence Data; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013997:Time Factors; D000098662:Vasodilator-Stimulated Phosphoprotein
D000470:Alkaloids; D002227:Carbazoles; D015815:Cell Adhesion Molecules; D018076:DNA, Complementary; D007211:Indoles; D008840:Microfilament Proteins; D010750:Phosphoproteins; D000098662:Vasodilator-Stimulated Phosphoprotein; C073601:KT 5823; D017869:Cyclic GMP-Dependent Protein Kinases; D006152:Cyclic GMP