Title: Development of an HTS assay for EPHX2 phosphatase activity and screening of nontargeted libraries.

Authors: Morisseau, Christophe; Sahdeo, Sunil; Cortopassi, Gino; Hammock, Bruce D

Published In Anal Biochem, (2013 Mar 01)

Abstract: The EPXH2 gene encodes soluble epoxide hydrolase (sEH), which has two distinct enzyme activities: epoxide hydrolase (Cterm-EH) and phosphatase (Nterm-phos). The Cterm-EH is involved in the metabolism of arachidonic acid epoxides that play important roles in blood pressure, cell growth, inflammation, and pain. While recent findings suggested complementary biological roles for Nterm-phos, research is limited by the lack of potent bioavailable inhibitors of this phosphatase activity. Also, a potent bioavailable inhibitor of this activity could be important in the development of therapy for cardiovascular diseases. We report herein the development of an HTS enzyme-based assay for Nterm-phos (Z'>0.9) using AttoPhos as the substrate. This assay was used to screen a wide variety of chemical entities, including a library of known drugs that have reached through clinical evaluation (Pharmakon 1600), as well as a library of pesticides and environmental toxins. We discovered that ebselen inhibits sEH phosphatase activity. Ebselen binds to the N-terminal domain of sEH (K(I)=550 nM) and chemically reacts with the enzyme to quickly and irreversibly inhibit Nterm-phos, and subsequently Cterm-EH, and thus represents a new class of sEH inhibitor.

PubMed ID: 23219563

MeSH Terms: Azoles/metabolism; Epoxide Hydrolases/analysis*; Epoxide Hydrolases/genetics; Epoxide Hydrolases/metabolism; High-Throughput Screening Assays*; Humans; Kinetics; Organoselenium Compounds/metabolism; Pesticides/metabolism; Protein Binding; Recombinant Proteins/analysis; Recombinant Proteins/biosynthesis; Recombinant Proteins/genetics; Small Molecule Libraries/chemistry*; Substrate Specificity; Toxins, Biological/metabolism