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 bridgeconstrained 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°C and pH 7.4 was ϳ500 nM. At the same conditions, the equilibrium dissociation constant K D , monitored by displacement of p-aminobenzamidine from the specificity pocket of uPA, was ϳ400 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.Serine proteases of the trypsin family (clan SA) have many physiological and pathophysiological functions. There is therefore extensive interest in generating specific inhibitors to be used for pharmacological interference with their enzymatic activity. Moreover, serine proteases are classical subjects for studies of catalytic and inhibitory mechanisms.Serine protease-catalyzed peptide bond hydrolysis proceeds through a tetrahedral transition state formed by a nucleophilic attack on the carbonyl group of the substrate P 1 amino acid by the hydroxyl group of Ser 195 (using the chymotrypsin template numbering (1)), with His 57 and Asp 102 acting as a charge relay system. The protonated His 57 functions as a general acid to facilitate collapse of the tetrahedral intermediate that is stabilized through interactions at the oxyanion hole and main chain ␤-strand-type hydrogen bonds between the P 1 -P 3 and P 2 Ј amino acids of the substrate and residues within the polypeptide binding cleft, as well as specific contacts within the S 1 , S 2 , S 3 , S 1 Ј, and S 2 Ј pockets, which bind respective side chains of the P 1 , P 2 , P 3 , P 1 Ј, and P 2 Ј residues (for reviews, see Refs. 2 and 3). Substrate specificity is governed by the fit of the P residues into their corresponding S-pockets as ...

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

“…As predicted from our previous report (Hansen et al, 2005), the modified sequence bound to human uPA with a K i indistinguishable from that of the original peptide: 35 Ϯ 8 M (n ϭ 5) for the modified sequence versus 30 Ϯ 1 M (n ϭ 3) for the original sequence. When planning which arginine analogs to test as the P1 residue in upain-2 and mupain-1, we considered varying several parameters, all expected to affect the interaction of the P1 residue with the S1 pocket ( Table 1).…”

“…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).…”

“…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.…”