Abstract
G protein coupled kinase type 4 (GRK4) reduces renal sodium excretion by deactivating the renal dopamine type 1 receptor (D 1 R) through serine phosphorylation. At least 3 polymorphisms in GRK4 have been shown to be associated with the expression of hypertension and/or salt sensitivity in humans and in mice. GRK4 is an ideal therapeutic target for treating hypertension since putting human GRK4 variants in mice results in hypertension which can be reversed by reducing the expression of GRK4. Inhibitors of GRK4 could serve as potent and selective agents to promote sodium excretion in salt sensitive and/or hypertensive individuals. Therefore, we developed an automated high throughput homogeneous time resolved fluorescent resonance energy transfer assay (TR-FRET) to identify small molecule inhibitors of GRK4 isoenzymes. The assay relies on the close proximity (90 A) transfer of 337 nm energy from a europium anti-phosphoserine antibody (Eu-pSer) to an allophycocyanin labeled streptavidin acceptor (streptavidin-APC), producing FRET at 665 nm. The assay was optimized through serial dilutions of streptavidin-APC, Eu-pSer, GRK4, and a peptide consisting of serine sites in the D 1 R, serving as a substrate. To validate the assay and determine its suitability for a large scale high throughput screen, three inhibitors, GRKA, GRKB, and GRKC were selected and concentration response assays were performed to determine IC50 values and variability analysis. The IC50 and (R 2 ) values of GRKA, GRKB, and GRKC were 4.99 μM (0.99), 26.52 μM (0.99), and 179.8 μM (0.82), respectively. GRKA is the most efficient GRK4 inhibitor, while GRKC is the least. This assay will be used to test congeners of compounds A, B, and C to identify more selective candidates, as well as for screening available small molecule compound libraries.
Published Version
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