title: Pluripotentialities of a quenched fluorescent peptide substrate library: enzymatic detection, characterization, and isoenzymes differentiation.
abstract: Protease inhibitors represent a major class of drugs, even though a large number of proteases remain unexplored. Consequently, a great interest lies in the identification of highly sensitive substrates useful for both the characterization and the validation of these enzyme targets and for the design of inhibitors as potential therapeutic agents through high-throughput screening (HTS). With this aim, a synthetic substrate library, in which the highly fluorescent (L)-pyrenylalanine residue (Pya) is efficiently quenched by its proximity with the p-nitro-(L)-phenylalanine (Nop) moiety, was designed. The cleavage between Pya and Nop leads to a highly fluorescent metabolite providing the required sensitivity. This library, characterized by a water-soluble primary sequence Ac-SGK-Pya-(X)(n)(-)Nop-GGK-NH(2), X being a mixture of 10 natural amino acids (A, I, L, K, F, W, E, Q, T, P) and n varying from 0 to 3, was validated using enzymes belonging to the four main types of hydrolases: serine-, metallo-, cystein-, and aspartyl-proteases. The selectivity of substrates belonging to this library was evidenced by characterizing specific substrates for the isoenzymes NEP-1 and NEP-2. This library easily synthesized is of great interest for the identification and development of selective and specific substrates for still uncharacterized endoproteases.
authors: Pharmaleads, Paris BioPark, 11 rue Watt 75013 Paris, France.
authors: Hervé
authors: H
authors: Poras
authors: Tanja
authors: T
authors: Ouimet
authors: Sou-Vinh
authors: SV
authors: Orng
authors: Emilie
authors: E
authors: Dangé
authors: Marie-Claude
authors: MC
authors: Fournié-Zaluski
authors: Bernard P
authors: BP
authors: Roques
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001671:Biochemistry / Q000379:methods*; D002853:Chromatography, Liquid; D006801:Humans; D006868:Hydrolysis; D007527:Isoenzymes / Q000378:metabolism; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010447:Peptide Hydrolases / Q000378:metabolism*; D019151:Peptide Library; D010455:Peptides / Q000737:chemistry / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D013379:Substrate Specificity
chemical_list: D007527:Isoenzymes; D019151:Peptide Library; D010455:Peptides; D010447:Peptide Hydrolases