A cyclic peptidylic inhibitor of murine urokinase-type plasminogen activator: Changing species specificity by substitution of a single residue
Résumé
Urokinase-type plasminogen activator (uPA) is a potential therapeutic target in a variety of pathological conditions, including cancer. In order to find new principles for inhibiting uPA in murine cancer models, we screened a phage-displayed peptide library with murine uPA as bait. We thereby isolated several murine uPA-binding peptide sequences, the most predominant of which was the disulphide-bridged constrained sequence CPAYSRYLDC, which we will refer to as mupain-1. A chemically synthesised peptide corresponding to this sequence was found to be a competitive inhibitor of murine uPA, inhibiting its activity towards a low molecular mass chromogenic substrate as well as against its natural substrate plasminogen. The Ki value for inhibition as well as the KD value for binding were around 400 nM. Among a variety of other murine and human serine proteases, including trypsin, mupain-1 was found to be highly selective for murine uPA and did not even inhibit human uPA measurably. The cyclic structure of mupain-1 was indispensable for binding. Alanine scanning mutagenesis identified R6 of mupain-1 as the P1 residue and indicated an extended binding interaction including the P5, P3, P2, P1, and P1‘ residues of mupain-1 and the specificity pocket, the catalytic triad, and the amino acids 41, 99 and 192 located in and around the active site of murine uPA. Exchanging histidine 99 of human uPA with tyrosine, the corresponding residue in murine uPA, conferred mupain-1 susceptibility onto the latter. Peptide derived inhibitors, such as mupain-1, may provide novel mechanistic information about enzyme-inhibitor interactions, provide alternative methodologies for designing effective protease inhibitors, and be used for target validation in murine model systems.
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