title: A sensitive fluorigenic substrate for selective in vitro and in vivo assay of leukotriene A4 hydrolase activity.
abstract: 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 (k(cat)/K(m)) of 3.8±0.5×10⁴ M⁻¹ s⁻¹ using human recombinant LTA4H and a limit of detection and quantification of less than 1 to 2 ng. 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.
authors.affiliation: Pharmaleads, Paris BioPark, 75013 Paris, France.
authors.forename: Hervé
authors.initials: H
authors.lastname: Poras
authors.forename: Sophie
authors.initials: S
authors.lastname: Duquesnoy
authors.forename: Marie-Claude
authors.initials: MC
authors.lastname: Fournié-Zaluski
authors.forename: Céline
authors.initials: C
authors.lastname: Ratinaud-Giraud
authors.forename: Bernard P
authors.initials: BP
authors.lastname: Roques
authors.forename: Tanja
authors.initials: T
authors.lastname: Ouimet
mesh_terms: D000818:Animals; D057075:Enzyme Assays / Q000379:methods*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004851:Epoxide Hydrolases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D008297:Male; D051379:Mice; D062105:Molecular Docking Simulation; D009281:Naphthalenes / Q000032:analysis / Q000378:metabolism*; D010455:Peptides / Q000032:analysis / Q000378:metabolism*; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013050:Spectrometry, Fluorescence / Q000379:methods; D013379:Substrate Specificity
chemical_list: D004791:Enzyme Inhibitors; D009281:Naphthalenes; D010455:Peptides; D011994:Recombinant Proteins; C072587:2-naphthylamide; D004851:Epoxide Hydrolases; C043134:leukotriene A4 hydrolase
keywords: Aminopeptidase; In vivo enzyme assay; LTA4 hydrolase; Specific inhibitor; Specific substrate