Elsevier

Analytical Biochemistry

Volume 343, Issue 1, 1 August 2005, Pages 66-75
Analytical Biochemistry

Fluorescent substrates for soluble epoxide hydrolase and application to inhibition studies

https://doi.org/10.1016/j.ab.2005.03.041Get rights and content

Abstract

Inhibition of the mammalian soluble epoxide hydrolase (sEH) is a promising new therapy in the treatment of disorders resulting from hypertension and vascular inflammation. A spectrophotometric assay (4-nitrophenyl-trans-2,3-epoxy-3-phenylpropyl carbonate, NEPC) is currently used to screen libraries of chemicals; however this assay lacks the required sensitivity to differentiate the most potent inhibitors. A series of fluorescent α-cyanoester and α-cyanocarbonate epoxides that produce a strong fluorescent signal on epoxide hydrolysis by both human and murine sEH were designed as potential substrates for an in vitro inhibition assay. The murine enzyme showed a broad range of specificities, whereas the human enzyme showed the highest specificity for cyano(6-methoxy-naphthalen-2-yl)methyl trans-[(3-phenyloxiran-2-yl)methyl] carbonate. An in vitro inhibition assay was developed using this substrate and recombinant enzyme. The utility of the fluorescent assay was confirmed by determining the IC50 values for a series of known inhibitors. The new IC50 values were compared with those determined by spectrophotometric NEPC and radioactive tDPPO assays. The fluorescent assay ranked these inhibitors on the basis of IC50 values, whereas the NEPC assay did not. The ranking of inhibitor potency generally agreed with that determined using the tDPPO assay. These results show that the fluorescence-based assay is a valuable tool in the development of sEH inhibitors by revealing structure–activity relationships that previously were seen only by using the costly and labor-intensive radioactive tDPPO assay.

Section snippets

Reagents

All reagents and solvents were purchased from Aldrich Chemical (Milwaukee, WI, USA) unless otherwise noted and were used without further purification. Triethylamine (TEA) was distilled over CaH2 prior to use. 4-Chlorocinnamyl alcohol was synthesized as per Charette et al. [27]. Hept-3-enoic acid (85%) was purchased from TCI Chemicals (Portland, OR, USA) and was used as received. Although not explicitly labeled as such, 1H NMR indicated greater than 85% trans-isomer when compared with an

Design and synthesis

The substrates shown in Table 1 were designed to follow the same mechanistic path of degradation as reported by Dietze et al. [19] for NEPC. It was necessary to find a fluorescent reporter group whose fluorescence was modulated on cleavage of the ester or carbonate linker. Although umbelliferone phenylepoxyesters have been reported to be good fluorescent substrates for sEH, they are hydrolytically unstable [19]. It has been shown that α-cyanoester derivatives of aldehyde 1 are essentially

Conclusion

We have developed a fluorescent assay for mammalian sEH inhibition studies. This assay is based on the enzymatic hydrolysis of a readily synthesized β-epoxy-carbonate that results in the production of a fluorescent aldehyde. This new assay has a sensitivity that is 100 times greater than that in the previously used spectrophotometric assay. This assay will be an invaluable tool for the development of new sEH inhibitors and will further our investigations into the relationship among sEH

Acknowledgments

The authors thank James Sanborn for many helpful discussions and Jozsef Lango for assistance with mass spectral determinations. We also thank Rong Zhang for project initiation. Paul Jones was supported by an NIH Institutional Pre- and Postdoctoral Training grant (T32 DK07355) and by an NIH/NHLBI Ruth L. Kirschstein–NRSA grant (F32 HL078096). Nicola Wolf was supported by the Bavarian Research Foundation (Bayerische Forschungsstiftung). This work was supported in part by NIEHS grant ES02710,

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