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

Christophe Morisseau, Sunil Sahdeo, Gino A Cortopassi, Bruce D. Hammock

Research output: Contribution to journalArticlepeer-review

17 Scopus citations

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 (KI = 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.

Original languageEnglish (US)
Pages (from-to)105-111
Number of pages7
JournalAnalytical Biochemistry
Volume434
Issue number1
DOIs
StatePublished - Mar 1 2013

Keywords

  • Ebselen
  • HTS assay
  • Phosphatase
  • Soluble epoxide hydrolase

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology
  • Cell Biology

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