A sensitive fluorigenic substrate for selective in vitro and in vivo assay of leukotriene A4 hydrolase activity.

Leukotriene A4 hydrolase (LTA4H) is a bifunctional zinc-dependent metalloprotease bearing both an epoxide hydrolase, producing the pro-inflammatory LTB4 leukotriene, and an aminopeptidase activity, whose physiological relevance has long been ignored. Distinct substrates are commonly used for each activity, although none is completely satisfactory; LTA4, substrate for the hydrolase activity, is unstable and inactivates the enzyme, whereas aminoacids β-naphthylamide and para-nitroanilide, used as aminopeptidase substrates, are poor and nonselective. Based on the three-dimensional structure of LTA4H, we describe a new, specific, and high-affinity fluorigenic substrate, PL553 [l-(4-benzoyl)phenylalanyl-β-naphthylamide], with both in vitro and in vivo applications. PL553 possesses a catalytic efficiency (kcat/Km) of 3.8±0.5×10(4)M(-1)s(-1) using human recombinant LTA4H and a limit of detection and quantification of less than 1 to 2ng. The PL553 assay was validated by measuring the inhibitory potency of known LTA4H inhibitors and used to characterize new specific amino-phosphinic inhibitors. The LTA4H inhibition measured with PL553 in mouse tissues, after intravenous administration of inhibitors, was also correlated with a reduction in LTB4 levels. This authenticates the assay as the first allowing the easy measurement of endogenous LTA4H activity and in vitro specific screening of new LTA4H inhibitors. Copyright © 2013 Elsevier Inc. All rights reserved.

Citation

Hervé Poras, Sophie Duquesnoy, Marie-Claude Fournié-Zaluski, Céline Ratinaud-Giraud, Bernard P Roques, Tanja Ouimet. A sensitive fluorigenic substrate for selective in vitro and in vivo assay of leukotriene A4 hydrolase activity. Analytical biochemistry. 2013 Oct 15;441(2):152-61

PMID: 23851339

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