Development of fluorescent substrates for microsomal epoxide hydrolase and application to inhibition studies

Abstract

The microsomal epoxide hydrolase (mEH) plays a significant role in the metabolism of numerous xenobiotics. Additionally, it has a potential role in sexual development and bile acid transport, and it is associated with a number of diseases, such as emphysema, spontaneous abortion, eclampsia and several forms of cancer. Toward developing chemical tools to study mEH biological role, we designed and synthesized a series of absorbent and fluorescent substrates. The highest activity for both rat and human mEH was obtained with the fluorescent substrate cyano(6-methoxy-naphthalen-2-yl)methyl glycidyl carbonate (11). An in vitro inhibition assay using this substrate ranked a series of known inhibitors similarly to the assay that used radioactive cis-stilbene oxide, but with a greater discrimination between inhibitors. These results demonstrate that the new fluorescence-based assay is a useful tool for the discovery of structure-activity relationships among mEH inhibitors. Further, this substrate could also be used for the screening chemical library with high accuracy and with a Z' value about 0.7. This new assay permits a significant decrease in labor and cost as well as offering the advantage of a continuous readout. However, it should not be used with crude enzyme preparations due to interfering reactions.

In Vitro Metabolism of Oprozomib, an Oral Proteasome Inhibitor: Role of Epoxide Hydrolases and Cytochrome P450s

“…An optimized protein concentration (2 μg/ml) and incubation time (30 min) were applied in the study to ensure the linearity over the course of the reaction with sufficient amounts of PR-176 formation for accurate quantification. As shown in Figure 3C were determined to be 56.9 ± 3.8 μM and 648 ± 17 nmol/min/mg proteins, respectively, which was comparable to previously reported literature values (Morisseau et al, 2011). Inhibition of oprozomib epoxide hydrolysis by a selective mEH inhibitor in HLMs.…”

“…Inhibition of oprozomib epoxide hydrolysis by a selective mEH inhibitor in HLMs. NSPA is a selective mEH inhibitor that has been previously characterized with recombinant rat and human mEH for its potency (Morisseau et al, 2008;Morisseau et al, 2011). Here, we evaluated its potency in inhibiting hydrolysis of cis-SO and oprozomib in HLMs.…”

“…The structures of these two inhibitors are shown in Supplemental Figure 1. Human recombinant microsomal epoxide hydrolase (mEH, # Cri 08.17, purity 80%) and soluble epoxide hydrolase (sEH, # Cri 71.16, purity 95%) were prepared and characterized as described previously (Morisseau et al, 2000;Morisseau et al, 2011). Supersomes containing cDNA-expressed human CYPs, CYP reductase and cytochrome b5 were purchased from BD Bioscience (San Jose, CA).…”

“…Recombinant human EHs were kept at -80 °C until use. Briefly, incubations (0.1 ml) were conducted in 0.1 M Tris-HCl buffer (pH 9.0 for mEH; pH 7.5 for sEH) containing 0.1 mg/ml fatty acid-free bovine serum albumin (BSA) (Morisseau et al, 2000;Morisseau et al, 2011). Enzyme activities of mEH and sEH (5 µg/ml) towards epoxide hydrolysis were confirmed using their corresponding probe substrates (50 µM) cis-stilbene oxide (cis-SO) and trans-stilbene oxide (trans-SO), respectively.…”

Inhibiting an Epoxide Hydrolase Virulence Factor from Pseudomonas aeruginosa Protects CFTR

“…[9] However, a parallel survey of xenobiotic substrates revealed that Cif can hydrolyze glycidyl methacrylate (Figure S5), the hydrolytic trigger in a collection of mono-substituted fluorogenic epoxides that was being developed in parallel for mammalian microsomal EHs. [13] Cif generated a robust hydrolysis signal with several members of the panel (Figure S6), and showed the highest activity against cyano(6-methoxynaphthalen-2-yl)methyl(oxiran-2-ylmethyl) (CMNGC, 2 ) ( Figure 3a ).…”