Development of an HTS assay for EPHX2 phosphatase activity and screening of nontargeted libraries.
Anal Biochem
; 434(1): 105-11, 2013 Mar 01.
Article
em En
| MEDLINE
| ID: mdl-23219563
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.
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Epóxido Hidrolases
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Bibliotecas de Moléculas Pequenas
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Ensaios de Triagem em Larga Escala
Tipo de estudo:
Diagnostic_studies
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Screening_studies
Limite:
Humans
Idioma:
En
Ano de publicação:
2013
Tipo de documento:
Article