title: Identification of an endothelin-converting enzyme-2-specific fluorigenic substrate and development of an in vitro and ex vivo enzymatic assay.
abstract: Endothelin-converting enzyme-2 (ECE-2) is a membrane-bound zinc-dependent metalloprotease that shares a high degree of sequence homology with ECE-1, but displays an acidic pH optimum characteristic of maturing enzymes acting late in the secretory pathway. Although ECE-2, like ECE-1, can cleave the big endothelin intermediate to produce the vasoconstrictive endothelin peptide, its true physiological function remains to be elucidated, a task that is hampered by the lack of specific tools to study and discriminate ECE-2 from ECE-1, i.e. specific substrates and/or specific inhibitors. To fill this gap, we searched for novel ECE-specific peptide substrates. To this end, peptides derived from the big endothelin intermediate were tested using ECE-1 and ECE-2, leading to the identification of an ECE-1-specific substrate. Moreover, screening of our proprietary fluorigenic peptide Fluofast® libraries using ECE-1 and ECE-2 allowed the identification of Ac-SKG-Pya-F-W-Nop-GGK-NH(2) (PL405), as a specific and high affinity ECE-2 substrate. Indeed, ECE-2 cleaved PL405 at the Pya-F amide bond with a specificity constant (k(cat)/K(m)) of 8.1 ± 0.9 × 10(3) M(-1) s(-1). Using this novel substrate, we also characterized the first potent (K(i) = 7.7 ± 0.3 nM) and relatively selective ECE-2 inhibitor and developed a quantitative fluorigenic ECE-2 assay. The assay was used to study the ex vivo ECE-2 activity in wild type and ECE-2 knock-out tissues and was found to truly reflect ECE-2 expression patterns. The PL405 assay is thus the first tool to study ECE-2 inhibition using high throughput screening or for ex vivo ECE-2 quantification.
authors.affiliation: Pharmaleads, Paris BioPark, 75013 Paris, France.
authors.forename: Tanja
authors.initials: T
authors.lastname: Ouimet
authors.forename: Sou-Vinh
authors.initials: SV
authors.lastname: Orng
authors.forename: Hervé
authors.initials: H
authors.lastname: Poras
authors.forename: Khatuna
authors.initials: K
authors.lastname: Gagnidze
authors.forename: Lakshmi A
authors.initials: LA
authors.lastname: Devi
authors.forename: Marie-Claude
authors.initials: MC
authors.lastname: Fournié-Zaluski
authors.forename: Bernard P
authors.initials: BP
authors.lastname: Roques
mesh_terms: D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000008:administration & dosage / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D000072396:Endothelin-Converting Enzymes; D016232:Endothelins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D057075:Enzyme Assays / Q000379:methods*; D004791:Enzyme Inhibitors / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry*; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology*; D006801:Humans; D008666:Metalloendopeptidases / Q000008:administration & dosage / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D051379:Mice; D009928:Organ Specificity; D019151:Peptide Library; D010455:Peptides / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D016232:Endothelins; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D019151:Peptide Library; D010455:Peptides; D016282:Aspartic Acid Endopeptidases; D008666:Metalloendopeptidases; C000606923:ECE1 protein, human; C000606924:ECE2 protein, human; C000606925:Ece1 protein, mouse; C000606926:Ece2 protein, mouse; D000072396:Endothelin-Converting Enzymes
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