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 journalArticle

15 Citations (Scopus)

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

Fingerprint

Epoxide Hydrolases
Phosphoric Monoester Hydrolases
Libraries
Assays
Screening
Epoxy Compounds
Blood pressure
Enzyme Assays
Enzyme activity
Cell growth
Enzymes
Pesticides
Arachidonic Acid
Metabolism
Blood Cells
Cardiovascular Diseases
Genes
Blood Pressure
Inflammation
Pain

Keywords

  • Ebselen
  • HTS assay
  • Phosphatase
  • Soluble epoxide hydrolase

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology
  • Cell Biology

Cite this

Development of an HTS assay for EPHX2 phosphatase activity and screening of nontargeted libraries. / Morisseau, Christophe; Sahdeo, Sunil; Cortopassi, Gino A; Hammock, Bruce D.

In: Analytical Biochemistry, Vol. 434, No. 1, 01.03.2013, p. 105-111.

Research output: Contribution to journalArticle

Morisseau, Christophe ; Sahdeo, Sunil ; Cortopassi, Gino A ; Hammock, Bruce D. / Development of an HTS assay for EPHX2 phosphatase activity and screening of nontargeted libraries. In: Analytical Biochemistry. 2013 ; Vol. 434, No. 1. pp. 105-111.
@article{5ee4db1ef6a047559edb67b4fd5ba7b9,
title = "Development of an HTS assay for EPHX2 phosphatase activity and screening of nontargeted libraries",
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.",
keywords = "Ebselen, HTS assay, Phosphatase, Soluble epoxide hydrolase",
author = "Christophe Morisseau and Sunil Sahdeo and Cortopassi, {Gino A} and Hammock, {Bruce D.}",
year = "2013",
month = "3",
day = "1",
doi = "10.1016/j.ab.2012.11.017",
language = "English (US)",
volume = "434",
pages = "105--111",
journal = "Analytical Biochemistry",
issn = "0003-2697",
publisher = "Academic Press Inc.",
number = "1",

}

TY - JOUR

T1 - Development of an HTS assay for EPHX2 phosphatase activity and screening of nontargeted libraries

AU - Morisseau, Christophe

AU - Sahdeo, Sunil

AU - Cortopassi, Gino A

AU - Hammock, Bruce D.

PY - 2013/3/1

Y1 - 2013/3/1

N2 - 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.

AB - 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.

KW - Ebselen

KW - HTS assay

KW - Phosphatase

KW - Soluble epoxide hydrolase

UR - http://www.scopus.com/inward/record.url?scp=84871977397&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84871977397&partnerID=8YFLogxK

U2 - 10.1016/j.ab.2012.11.017

DO - 10.1016/j.ab.2012.11.017

M3 - Article

C2 - 23219563

AN - SCOPUS:84871977397

VL - 434

SP - 105

EP - 111

JO - Analytical Biochemistry

JF - Analytical Biochemistry

SN - 0003-2697

IS - 1

ER -