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 (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.
authors.affiliation: Department of Entomology and UCD Comprehensive Cancer Center, CA 95616, USA. chmorisseau@ucdavis.edu
authors.forename: Christophe
authors.initials: C
authors.lastname: Morisseau
authors.forename: Sunil
authors.initials: S
authors.lastname: Sahdeo
authors.forename: Gino
authors.initials: G
authors.lastname: Cortopassi
authors.forename: Bruce D
authors.initials: BD
authors.lastname: Hammock
mesh_terms: D001393:Azoles / Q000378:metabolism; D004851:Epoxide Hydrolases / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D057166:High-Throughput Screening Assays; D006801:Humans; D054833:Isoindoles; D007700:Kinetics; D016566:Organoselenium Compounds / Q000378:metabolism; D010575:Pesticides / Q000378:metabolism; D011485:Protein Binding; D011994:Recombinant Proteins / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics; D054852:Small Molecule Libraries / Q000737:chemistry*; D013379:Substrate Specificity; D014118:Toxins, Biological / Q000378:metabolism
chemical_list: D001393:Azoles; D054833:Isoindoles; D016566:Organoselenium Compounds; D010575:Pesticides; D011994:Recombinant Proteins; D054852:Small Molecule Libraries; D014118:Toxins, Biological; C042986:ebselen; D004851:Epoxide Hydrolases; C506428:EPHX2 protein, human