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A High Throughput Screen for Biomining Cellulase Activity from Metagenomic Libraries
Published on: February 1, 2011
Development of a high throughput cell-based assay for soluble epoxide hydrolase using BacMam technology
Wensheng Xie1, Xiaoyan Tang, Quinn Lu
1Biological Reagents and Assay Development, Molecular Discovery Research, GlaxoSmithKline, 1250 S. Collegeville Road, Collegeville, PA 19426, USA.
Molecular Biotechnology
|March 27, 2010
Summary
Researchers developed a novel high-throughput assay to screen for soluble epoxide hydrolase (sEH) inhibitors. This fluorescence polarization assay effectively measures sEH activity and identifies potential drug candidates for cardiovascular diseases.
Area of Science:
- Biochemistry
- Pharmacology
- Assay Development
Background:
- Epoxyeicosatrienoic acids (EETs) offer cardiovascular and renal protection.
- Soluble epoxide hydrolase (sEH) rapidly inactivates EETs.
- Inhibiting sEH to boost EET levels is a promising therapeutic strategy for cardiovascular conditions.
Purpose of the Study:
- To develop a high-throughput, cell-based assay for measuring sEH activity.
- To screen small molecular compounds as potential sEH inhibitors.
- To validate the assay's performance against established methods.
Main Methods:
- Utilized fluorescence polarization (FP) technology.
- Employed a Cy3B-labeled 14,15-DHET ligand and a rabbit anti-14,15-DHET antibody.
- Assayed sEH activity in endogenous and engineered cell lines (HEK-293).
Main Results:
- Successfully developed and optimized a cell-based FP assay for sEH activity.
- Demonstrated good agreement between FP assay results and a commercial ELISA kit (pIC50 values).
- Evaluated known sEH inhibitors in sEH BacMam transduced HEK-293 cells.
Conclusions:
- The developed FP assay is the first high-throughput, cell-based method for screening sEH inhibitors.
- This assay provides a robust platform for identifying novel therapeutics targeting sEH.
- The findings support the therapeutic potential of sEH inhibition in cardiovascular diseases.
