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Proceedings of the 31 st European Peptide SymposiumMichal Lebl, Morten Meldal, Knud J. Jensen, Thomas Hoeg-Jensen (Editors)European Peptide Society, 2010Specific Substrates and Inhibitors of Human FurinAdam Lesner, Magdalena Wysocka, Anna Łęgowska,and Krzysztof RolkaUniversity of Gdansk, Faculty of Chemistry, Sobieskiego 18, 80-952, Gdansk, PolandIntroductionProprotein convertases (PCs) are calcium-dependent serine proteases of the subtilisinfamily which are known to generate the important biologically active peptides by thespecific cleavage of peptide bond at the C-terminal fragments R-X-(K/R)-R of theirprecursors. Such groups of enzymes are initially synthesized as inactive proenzyme formsthat undergo activation under physiological conditions. Furin, one of the sevenendoproteases that belong to the PC family, is ubiquitously expressed in mammalian cellsprocessing and activating a wide variety of proprotein substrates. The endogenoussubstrates of furin are receptors such as insulin proreceptor, plasma proteins, growth factorsand hormones as well as proteases such as matrix metalloproteinases – like membrane type1-matrix metalloproteinase (MT1-MMP). Increased activity of furin was identified in headand breast tumors, neck tumors, and also in lung cancer. Recent reports have indicated thatfurin is a target of hypoxia-inducible factor 1 (HIF-1) and that hypoxia induces elevatedlevels of furin, leading to more aggressive tumors. Therefore PC inhibitors may constitutenew promising antitumor agents. The aim of this study is to select, using combinatorialchemistry methods, specific furin substrates that will display FRET and chromogenicproperties. The general formula of the peptide library synthesized is as follows:where:ABZ-X 4 -X 3 -X 2 -Arg-ANB-NH 2 ,ABZ = 2-aminobenzoic acid (donor of fluorescence);ANB-NH 2 = amide of 5-amino-2–nitrobenzoic acid (acceptor of fluorescence);X 2 – X 4 = nonproteinogenic basic amino acids (D-Arg, Amf, Gnf, Har, Orn, Dab, lys)along with Arg and Ala (used as reference amino acids) were introduced and their chemicalstructure are displayed on Figure 1.Fig. 1. Chemical formulas of nonproteinogenic basic amino acids used to construct thepeptide library.Results and DiscussionIterative method was applied for selection process of the most efficiently hydrolyzedsubstrates. Among non proteinogenic basic charged residues present in the library X 3position which corresponds to the substrate P 4 position, the most efficient fluorescence was198