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.
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.
Hansen
Martin
M
Department of Molecular Biology, University of Aarhus, Gustav Wied's Vej 10C, 8000 Aarhus C, Denmark.
Wind
Troels
T
Blouse
Grant E
GE
Christensen
Anni
A
Petersen
Helle H
HH
Kjelgaard
Signe
S
Mathiasen
Lisa
L
Holtet
Thor L
TL
Andreasen
Peter A
PA
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
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