期刊
ANALYTICAL BIOCHEMISTRY
卷 343, 期 1, 页码 66-75出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ab.2005.03.041
关键词
soluble epoxide hydrolase; alpha-cyanoester; alpha-cyanocarbonate; kinetic assay; fluorescent substrate
资金
- NHLBI NIH HHS [F32 HL 078096, R41 HL 078016, F32 HL078096, R41 HL078016] Funding Source: Medline
- NIDDK NIH HHS [T32 DK007355, T32 DK 07355] Funding Source: Medline
- NIEHS NIH HHS [R37 ES002710, R01 ES002710, P42 ES004699, ES 02710, P42 ES 04699] Funding Source: Medline
- NINDS NIH HHS [R03 NS050841, R03 NS 050841] Funding Source: Medline
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 alpha-cyanoester and alpha-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. (c) 2005 Elsevier Inc. All rights reserved.
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