A cyclic peptidylic inhibitor of murine urokinase-type plasminogen activator: changing species specificity by substitution of a single residue

Abstract

uPA (urokinase-type plasminogen activator) 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 predominant of which was the disulfide-bridged constrained sequence CPAYSRYLDC, which we will refer to as mupain-1. A chemically synthesized 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 towards its natural substrate plasminogen. The K(i) value for inhibition as well as the K(D) value for binding were approx. 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 measurably inhibit human uPA. The cyclic structure of mupain-1 was indispensable for binding. Alanine scanning mutagenesis identified Arg(6) 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 amino acids 41, 99 and 192 located in and around the active site of murine uPA. Exchanging His(99) of human uPA by a tyrosine residue, the corresponding residue in murine uPA, conferred mupain-1 susceptibility on to 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.

Elucidation of the Contribution of Active Site and Exosite Interactions to Affinity and Specificity of Peptidylic Serine Protease Inhibitors Using Non-Natural Arginine Analogs

“…Thus, upain-1 binds approximately 500-fold better to human uPA than to murine uPA, whereas mupain-1 binds more than 2000-fold better to murine uPA than to human uPA. Both bind very selectively to uPA compared with other serine proteases from the same species (Hansen et al, 2005;Andersen et al, 2008). Analysis of the inhibitory mechanism showed that both upain-1 and mupain-1 are competitive inhibitors of their target enzymes (Hansen et al, 2005;Andersen et al, 2008).…”

“…Two-chain murine uPA was purchased from Molecular Innovations (Novi, MI). Wild-type and mutant recombinant human and murine uPA were expressed in and in some cases purified from human embryonic kidney 293T cells transfected with the corresponding cDNAs in pcDNA3.1 (Petersen et al, 2001;Andersen et al, 2008).…”

“…Both bind very selectively to uPA compared with other serine proteases from the same species (Hansen et al, 2005;Andersen et al, 2008). Analysis of the inhibitory mechanism showed that both upain-1 and mupain-1 are competitive inhibitors of their target enzymes (Hansen et al, 2005;Andersen et al, 2008). Site-directed mutagenesis (Hansen et al, 2005;Andersen et al, 2008) and X-ray crystal structure analysis (Zhao et al, 2007) showed that Arg4 of upain-1 and Arg6 of mupain-1 are the P1 1 residues of these peptides, i.e., are inserted into the enzymes' S1 or specificity pocket, the primary interaction site between substrate and enzyme.…”

“…Analysis of the inhibitory mechanism showed that both upain-1 and mupain-1 are competitive inhibitors of their target enzymes (Hansen et al, 2005;Andersen et al, 2008). Site-directed mutagenesis (Hansen et al, 2005;Andersen et al, 2008) and X-ray crystal structure analysis (Zhao et al, 2007) showed that Arg4 of upain-1 and Arg6 of mupain-1 are the P1 1 residues of these peptides, i.e., are inserted into the enzymes' S1 or specificity pocket, the primary interaction site between substrate and enzyme. In the case of upain-1, X-ray crystal structure analysis showed that the side-chain of the peptide's Glu7 blocks the enzyme's oxyanion hole (Zhao et al, 2007).…”

“…In particular, uPA is central to the invasive capacity of malignant tumors (for review, see Andreasen et al, 2000). We previously isolated two peptides, one (upain-1, CSWRGLENHRMC) being an inhibitor of human uPA (Hansen et al, 2005) and the other (mupain-1, CPAYSRYLDC) being an inhibitor of murine uPA (Andersen et al, 2008). Both are constrained in a circular form by a disulfide bridge.…”