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Development of a High Throughput Cell-Based Assay for Soluble Epoxide Hydrolase Using BacMam Technology

Author:
Xie, Wensheng, Tang, Xiaoyan, Lu, Quinn, Ames, Robert S., Ratcliffe, Steven J., Li, Hu
Source:
Molecular biotechnology 2010 v.45 no.3 pp. 207-217
ISSN:
1073-6085
Subject:
acids, antibodies, cardiovascular diseases, enzyme-linked immunosorbent assay, epoxide hydrolase, fluorescence, rabbits, screening
Abstract:
Epoxyeicosatrienoic acids (EETs) play important protective functions in cardiovascular and renal systems. Under physiological conditions, EETs are quickly converted by the soluble epoxide hydrolase (sEH) to diols which do not have the beneficiary roles. Inhibition of sEH with small molecules to increase the concentration of EETs therefore provides an attractive therapeutic strategy for cardiovascular diseases. We describe here the development of a high throughput cell-based assay to measure sEH activity and screen small molecular compounds as sEH inhibitors. This assay is based on the technology of fluorescence polarization (FP), utilizing a Cy3B labeled 14,15-DHET ligand and a rabbit anti-14,15-DHET antibody. With the optimized assay, we measured the cellular sEH activity of several cell lines expressing endogenous sEH as well as sEH BacMam transduced HEK-293 cells. The inhibitory effect of several known sEH inhibitors was evaluated in sEH BacMam transduced HEK-293 cells. Our data show that there is good agreement of pIC₅₀ values obtained between the FP format and a commercially available ELISA kit. To our knowledge, this is the first report of a high throughput cell-based assay for screening sEH inhibitors.
Agid:
2250443