Förster resonance energy transfer competitive displacement assay for human soluble epoxide hydrolase

Abstract

The soluble epoxide hydrolase (sEH), responsible for the hydrolysis of various fatty acid epoxides to their corresponding 1,2-diols, is becoming an attractive pharmaceutical target. These fatty acid epoxides, particularly epoxyeicosatrienoic acids (EETs), play an important role in human homeostatic and inflammation processes. Therefore, inhibition of human sEH, which stabilizes EETs in vivo, brings several beneficial effects to human health. Although there are several catalytic assays available to determine the potency of sEH inhibitors, measuring the in vitro inhibition constant (Ki) for these inhibitors using catalytic assay is laborious. In addition, koff, which has been recently suggested to correlate better with the in vivo potency of inhibitors, has never been measured for sEH inhibitors. To better measure the potency of sEH inhibitors, a reporting ligand, 1-(adamantan-1-yl)-3-(1-(2-(7-hydroxy-2-oxo-2H-chromen-4-yl)acetyl) piperidin-4-yl)urea (ACPU), was designed and synthesized. With ACPU, we have developed a Förster resonance energy transfer (FRET)-based competitive displacement assay using intrinsic tryptophan fluorescence from sEH. In addition, the resulting assay allows us to measure the Ki values of very potent compounds to the picomolar level and to obtain relative koff values of the inhibitors. This assay provides additional data to evaluate the potency of sEH inhibitors.

Lipoxin Generation Is Related to Soluble Epoxide Hydrolase Activity in Severe Asthma

“…These sEHIs are tight binding inhibitors that approach stoichiometric interaction with sEH. They are competitive and reversible inhibitors that have high target occupancy and a slow off rate (37). The consistent findings with all four compounds indicate a class effect for sEH inhibition, rather than an off-target action of a single compound.…”

“…The consistent findings with all four compounds indicate a class effect for sEH inhibition, rather than an off-target action of a single compound. Detailed information on the synthesis, physical properties, and pharmacokinetics of each of these sEHIs has been published (22,(35)(36)(37). Together, these findings support sEH inhibition as a new pharmacologic mechanism for enhancing endogenous LX generation in SA.…”

Symmetric adamantyl-diureas as soluble epoxide hydrolase inhibitors

“…Because of this, determination of residence time, the period when the drug is bound to enzyme, is very important for predicting biological activity. 26 Interestingly, among the 4 compounds tested, the k off were distributed over a 5-fold difference range and parallel the IC 50 s. These data indicate that spacers between the urea group and the the adamantane lead to a slower k off and have an optimal length. These results suggest that the molecular flexibility given by the alkyl spacers allows a better fit to the enzyme’s active site, including formation of a new hydrogen bond by the second urea group.…”

“…26 Compounds with the same molecular weight (two adamantane groups, two urea groups and eight carbons comprising various spacers between those groups) were selected for further testing to ensure that any difference arises from specific structural feature and not due to a general effect of the molecules.…”

“…These values are very similar and are almost equal with the variation of the assay over a narrow range of K i s. Because the urea inhibitors are slow-tight binding ligands of sEH, the IC 50 s measured after only 5 minutes incubation, as shown here, probably underestimated the true potency of some compounds, explaining the apparent divergence between IC 50 and K i values. 26 The K i is a ratio of kinetic off and on rates which are independently altered by structure. K i reflects the strength of the interaction between the inhibitor and the sEH.…”