Human APJ is a 7-transmembrane domain, Gai-coupled receptor (GPCR) that shares considerable sequence similarity with the angiotensin II receptor (AT-1) gene. Despite the similarities, APJ does not bind for angiotensin II, and was classified as an orphan receptor until the discovery of its ligand, apelin. The Apelin/APJ system has been demonstrated to elicit a variety of physiological effects on the heart and blood vessels, hypothalamus, and the endocrine system, but the role of apelin and APJ in vivo remains controversial. Adding to the questionable physiologic relevance is the report that APJ-/- mice exhibit no phenotype. The nascent study of apelin/APJ would benefit from a chemical probe to determine the precise role of apelin/APJ in vivo. To address this need, we propose to develop a functional assay to identify compounds that act as reversible antagonists at the human APJ-receptor.
In aim 1, we will construct stable cells lines expressing human APJ to be used in two complimentary cell-based assays. These assays will then be used to interrogate chemical libraries for molecules that modulate the function of APJ. The first assay utilizes EVX-1LUC, a cAMP sensitive luciferase reporter gene. In preliminary studies, APJ repressed forskolin (15 mu M) stimulated EVX- 1LUC in transiently transfected HEK293T cells by 40-fold (45,910+/-7237 vs. 10,186+/-1096 Light units). This robust result is consistent with APJ acting as a Gai-PCR to decrease intracellular cAMP. The second assay uses the commercially available Lance homogeneous time-resolved fluorescence resonance energy transfer (TR-FRET) assay to measure cAMP accumulation. Together these complimentary assay platforms will be used to confirm hits and eliminate format specific artifacts. A counter-screen against another Gai-coupled receptor unrelated to APJ (opiate receptor-like-1) has already been developed at Scripps and will be used to validate the specificity of results from the primary and secondary assays.
In aim 2, the optimized assays will be screened using the Sigma LOPAC small compound collection (n =1280). The results of this effort will formally establish the parameters of signal to noise, CV, and Z' factor and 'hit' rate and demonstrate the feasibility of our screening paradigm for a full MLSCN screen. Successful completion of the proposed research will provide a tool for additional functional studies to determine the precise role of apelin/APJ in cardiovascular disease, obesity and insulin resistance, and HIV infection and help to evaluate the potential of this receptor system as a future drug target. The recently discovered Apelin/APJ system has been reported to play a role in heart disease, obesity, and HIV infection. Despite these findings, the mechanistic roles of apelin and APJ in physiology and pathology remain to be determined. Our goal is to identify antagonists of apelin/APJ. Successful completion of the proposed research will provide a tool for additional studies to determine the precise role of apelin/APJ in cardiovascular regulation, obesity and insulin resistance, and HIV infection and help to evaluate the potential of this receptor system as a future drug target. ?
| Maloney, Patrick R; Khan, Pasha; Hedrick, Michael et al. (2012) Discovery of 4-oxo-6-((pyrimidin-2-ylthio)methyl)-4H-pyran-3-yl 4-nitrobenzoate (ML221) as a functional antagonist of the apelin (APJ) receptor. Bioorg Med Chem Lett 22:6656-60 |
| Siddiquee, Khandaker; Hampton, Jessica; Khan, Susan et al. (2011) Apelin protects against angiotensin II-induced cardiovascular fibrosis and decreases plasminogen activator inhibitor type-1 production. J Hypertens 29:724-31 |
