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

Anal Biochem. 2013 Mar 1;434(1):105-11. doi: 10.1016/j.ab.2012.11.017. Epub 2012 Dec 3.

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

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

Substances

  • Azoles
  • Organoselenium Compounds
  • Pesticides
  • Recombinant Proteins
  • Small Molecule Libraries
  • Toxins, Biological
  • ebselen
  • Epoxide Hydrolases
  • EPHX2 protein, human