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A small fluorescent ligand to investigate soluble epoxide hydrolase engagement in living cells

Affiliation
Institute of Pharmaceutical Chemistry, Goethe University Frankfurt, Max-von-Laue-Str. 9, 60438 Frankfurt am Main, Germany
Schönfeld, Julia;
Affiliation
Institute of Pharmaceutical Chemistry, Goethe University Frankfurt, Max-von-Laue-Str. 9, 60438 Frankfurt am Main, Germany
Brunst, Steffen;
Affiliation
Institute of Pharmaceutical Biology, Goethe University Frankfurt, Max-von-Laue-Str. 9, 60438 Frankfurt am Main, Germany
Burgers, Luisa;
Affiliation
Buchmann Institute for Molecular Life Sciences and Institute for Cell Biology and Neuroscience (BMLS), Max-von-Laue-Str. 15, 60438 Frankfurt am Main, Germany
Breunig, Peter;
Affiliation
Buchmann Institute for Molecular Life Sciences and Institute for Cell Biology and Neuroscience (BMLS), Max-von-Laue-Str. 15, 60438 Frankfurt am Main, Germany
DeMeglio, Murphy;
Affiliation
Institute of Pharmaceutical Chemistry, Goethe University Frankfurt, Max-von-Laue-Str. 9, 60438 Frankfurt am Main, Germany
Ehrler, Johanna H. M.;
Affiliation
Institute of Pharmaceutical Chemistry, Goethe University Frankfurt, Max-von-Laue-Str. 9, 60438 Frankfurt am Main, Germany
Lillich, Felix F.;
Affiliation
Institute of Pharmaceutical Chemistry, Goethe University Frankfurt, Max-von-Laue-Str. 9, 60438 Frankfurt am Main, Germany
Weizel, Lilia;
Affiliation
Institute of Pharmaceutical Biology, Goethe University Frankfurt, Max-von-Laue-Str. 9, 60438 Frankfurt am Main, Germany
Fürst, Robert;
Affiliation
Buchmann Institute for Molecular Life Sciences and Institute for Cell Biology and Neuroscience (BMLS), Max-von-Laue-Str. 15, 60438 Frankfurt am Main, Germany
Hefendehl, Jasmin;
Affiliation
Institute of Pharmaceutical Chemistry, Goethe University Frankfurt, Max-von-Laue-Str. 9, 60438 Frankfurt am Main, Germany
Proschak, Ewgenij;
Affiliation
Institute of Pharmaceutical Chemistry, Goethe University Frankfurt, Max-von-Laue-Str. 9, 60438 Frankfurt am Main, Germany
Hiesinger, Kerstin

Soluble epoxide hydrolase (sEH) is ubiquituos in the human organism and metabolizes highly biologically active epoxy-fatty acids (EETs) to the less active corresponding diols. Since sEH metabolism is the main pathway for the deactivation of EETs it represents a promising target for a variety of diseases.  In disease models of central nervous system disorders such as Alzheimer´s or Parkinson´s disease sEH upregulation was observed while inhibition of sEH resulted in neuroprotective effects. In this work, we present the design and synthesis of the small fluorescent sEH ligand JSF68. Starting from the potent sEH inhibitor GSK2256294A we replaced the triazine moiety by the small fluorophore nitrobenzoxadiazole (NBD). The resulting fluorescent sEH ligand displayed excellent potency in vitro (IC50 < 1.5 nM in our enzyme activity assay). We demonstrate the applicability of JSF68 in a NanoBRET-based assay which is the first sEH target engagement assay in living cells reported so far. Furthermore, the fluorescent ligand was used to detect sEH in sEH-transfected HEK293T cells and in primary mouse astrocytes by fluorescence microscopy.

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