title: A urokinase-type plasminogen activator-inhibiting cyclic peptide with an unusual P2 residue and an extended protease binding surface demonstrates new modalities for enzyme inhibition.
abstract: To find new principles for inhibiting serine proteases, we screened phage-displayed random peptide repertoires with urokinase-type plasminogen activator (uPA) as the target. The most frequent of the isolated phage clones contained the disulfide bridge-constrained sequence CSWRGLENHRMC, which we designated upain-1. When expressed recombinantly with a protein fusion partner, upain-1 inhibited the enzymatic activity of uPA competitively with a temperature and pH-dependent K(i), which at 25 degrees C and pH 7.4 was approximately 500 nm. At the same conditions, the equilibrium dissociation constant K(D), monitored by displacement of p-aminobenzamidine from the specificity pocket of uPA, was approximately 400 nm. By an inhibitory screen against other serine proteases, including trypsin, upain-1 was found to be highly selective for uPA. The cyclical structure of upain-1 was indispensable for uPA binding. Alanine-scanning mutagenesis identified Arg(4) of upain-1 as the P(1) residue and indicated an extended binding interaction including the specificity pocket and the 37-, 60-, and 97-loops of uPA and the P(1), P(2), P(3)', P(4)', and the P(5)' residues of upain-1. Substitution with alanine of the P(2) residue, Trp(3), converted upain-1 into a distinct, although poor, uPA substrate. Upain-1 represents a new type of uPA inhibitor that achieves selectivity by targeting uPA-specific surface loops. Most likely, the inhibitory activity depends on its cyclical structure and the unusual P(2) residue preventing the scissile bond from assuming a tetrahedral geometry and thus from undergoing hydrolysis. Peptide-derived inhibitors such as upain-1 may provide novel mechanistic information about enzyme-inhibitor interactions and alternative methodologies for designing effective protease inhibitors.
authors.affiliation: Department of Molecular Biology, University of Aarhus, Gustav Wied's Vej 10C, 8000 Aarhus C, Denmark.
authors.forename: Martin
authors.initials: M
authors.lastname: Hansen
authors.forename: Troels
authors.initials: T
authors.lastname: Wind
authors.forename: Grant E
authors.initials: GE
authors.lastname: Blouse
authors.forename: Anni
authors.initials: A
authors.lastname: Christensen
authors.forename: Helle H
authors.initials: HH
authors.lastname: Petersen
authors.forename: Signe
authors.initials: S
authors.lastname: Kjelgaard
authors.forename: Lisa
authors.initials: L
authors.lastname: Mathiasen
authors.forename: Thor L
authors.initials: TL
authors.lastname: Holtet
authors.forename: Peter A
authors.initials: PA
authors.lastname: Andreasen
mesh_terms: D010129:4-Aminobenzoic Acid / Q000737:chemistry; D000409:Alanine / Q000737:chemistry; D000595:Amino Acid Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D036022:Capsid Proteins; D002384:Catalysis; D002460:Cell Line; D004247:DNA / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay; D015951:Factor Xa / Q000737:chemistry; D006801:Humans; D006863:Hydrogen-Ion Concentration; D020128:Inhibitory Concentration 50; D007700:Kinetics; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010447:Peptide Hydrolases / Q000737:chemistry; D019151:Peptide Library; D010455:Peptides / Q000737:chemistry; D010456:Peptides, Cyclic / Q000737:chemistry* / Q000502:physiology; D010958:Plasminogen / Q000737:chemistry; D011480:Protease Inhibitors / Q000494:pharmacology; D011485:Protein Binding; D011486:Protein C / Q000737:chemistry; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D013379:Substrate Specificity; D013696:Temperature; D013816:Thermodynamics; D013997:Time Factors; D014357:Trypsin / Q000737:chemistry; D020298:U937 Cells; D014568:Urokinase-Type Plasminogen Activator / Q000737:chemistry*; D014760:Viral Fusion Proteins / Q000737:chemistry
chemical_list: D036022:Capsid Proteins; D004268:DNA-Binding Proteins; D004220:Disulfides; D004791:Enzyme Inhibitors; D019151:Peptide Library; D010455:Peptides; D010456:Peptides, Cyclic; D011480:Protease Inhibitors; D011486:Protein C; D011994:Recombinant Proteins; D014760:Viral Fusion Proteins; C506777:upain-1 peptide; D010958:Plasminogen; D004247:DNA; D010447:Peptide Hydrolases; D014357:Trypsin; D015951:Factor Xa; D014568:Urokinase-Type Plasminogen Activator; D000409:Alanine; D010129:4-Aminobenzoic Acid