Identification of novel prostate‐specific antigen‐binding peptides modulating its enzyme activity

Wu, Ping; Leinonen, Jari; Koivunen, Erkki; Lankinen, Hilkka; Stenman, Ulf‐Hǎkan

doi:10.1046/j.1432-1327.2000.01696.x

Cited by 71 publications

(57 citation statements)

References 46 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Libraries with disulfide bridge-constrained peptides were previously used to isolate an activity-enhancing, prostate-specific antigen-binding peptide sequence (36,37) and an inhibitory chymotrypsin-binding peptide sequence with a micromolar K i value (38). The peptide sequence described here is a competitive inhibitor of the enzymatic activity of uPA, inhibiting its plasminogen activation activity as well as its peptidolytic activity.…”

Section: Discussionmentioning

confidence: 99%

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

Hansen

Wind

Blouse

et al. 2005

Journal of Biological Chemistry

View full text Add to dashboard Cite

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 ...

show abstract

Section: Discussionmentioning

confidence: 99%

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

Hansen

Wind

Blouse

et al. 2005

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…This may be explained by the fact that some of the GST fusion proteins occur as dimers and trimers, which facilitate multivalent binding and enhanced avidity (12 ). Even so, the IPMA is not sensitive enough for measurement of active PSA in sera with the moderately increased concentrations that are typically present at early stages of PCa and BPH.…”

Section: Discussionmentioning

confidence: 99%

“…PSA-binding peptides were identified by screening phage display peptide libraries (12 ). Two peptides, B-2 and C-4, were constructed as GST fusion proteins, expressed in Escherichia coli BL 21 cells and purified by glutathione affinity chromatography (Amersham Pharmacia Biotech) as described previously (12 ).…”

Section: Psa-binding Peptidesmentioning

confidence: 99%

See 1 more Smart Citation

Immunopeptidometric Assay for Enzymatically Active Prostate-Specific Antigen

Zhu

Stenman

et al. 2004

Clinical Chemistry

Self Cite

View full text Add to dashboard Cite

Background: Determinations of certain forms of prostate-specific antigen (PSA) have been shown to increase the specificity for prostate cancer (PCa). One such variant, proteolytically active PSA, is a potentially useful tumor marker, but it is not specifically recognized by antibodies. Using phage display libraries, we previously identified a "family" of peptides that bind specifically to active PSA. We used these to develop an immunopeptidometric assay (IPMA) that specifically detects this form of PSA. Methods: Microtitration plates coated with a PSA antibody were used to capture PSA, and a PSA-binding glutathione S-transferase (GST) fusion peptide was used as a tracer. Bound tracer was detected with an antibody to GST labeled with a europium chelate. PSA isoenzymes with high and low enzymatic activity were used to study binding specificity. Results: The IPMA detected enzymatically active PSA but not internally cleaved PSA and pro-PSA, which are enzymatically inactive. The assay detected 1-10% of free PSA in serum from PCa patients. Conclusions: Peptides identified by phage display can be used to develop assays with unique specificities for enzymatically active PSA. IPMA represents a new assay principle with wide potential utility.

show abstract

“…Development of small molecule inhibitors has proved to be a daunting task (for review, see Rockway et al, 2002). To develop inhibitors with better specificity, a number of peptidylic serine protease inhibitors have recently been isolated by screening phage-displayed peptide libraries, including inhibitors of chymotrypsin (Krook et al, 1997(Krook et al, -1998, factor VIIa (Dennis et al, 2000), prostate-specific antigen (Wu et al, 2000), kallikrein-2 (Hekim et al, 2006;Heinis et al, 2009), mannose-binding lectin-associated serine protease-1 and -2 (Kocsis et al, 2010), furins (Hajdin et al, 2010), and isoforms of trypsin (Wu et al, 2010).…”

Section: Introductionmentioning

confidence: 99%

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

Hosseini

Jiang

Sørensen³

et al. 2011

Mol Pharmacol

View full text Add to dashboard Cite

There is increasing interest in developing peptides for pharmacological intervention with pathophysiological functions of serine proteases. From phage-displayed peptide libraries, we previously isolated peptidylic inhibitors of urokinase-type plasminogen activator, a potential target for intervention with cancer invasion. The two peptides, upain-1 (CSWRGLENHRMC) and mupain-1 (CPAYSRYLDC), are competitive inhibitors of human and murine urokinase-type plasminogen activator, respectively. Both have an Arg as the P1 residue, inserting into the S1 pocket in the active site of the enzymes, but their specificity depends to a large extent on interactions outside the enzymes' active sites, so-called exosite interactions. Here we describe upain-2 (CSWRGLENHAAC) and the synthesis of a number of upain-2 and mupain-1 variants in which the P1 Arg was substituted with novel non-natural Arg analogs and achieved considerable improvement in the affinity of the peptides to their targets. Using chimeras of human and murine urokinase-type plasminogen activator as well as X-ray crystallography, we delineated the relative contribution of the P1 residue and exosite interactions to the affinity and specificity of the inhibitors for their target enzyme. The effect of inserting a particular non-natural amino acid into the P1 position is determined by the fact that changes in interactions of the P1 residue in the S1 pocket lead to changed exosite interactions and vice versa. These findings are of general interest when the affinities and specificities of serine protease inhibitors to be used for pharmacological intervention are considered and could pave the way for potential drug candidates for the treatment of cancer.

show abstract

Identification of novel prostate‐specific antigen‐binding peptides modulating its enzyme activity

Cited by 71 publications

References 46 publications

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

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

Immunopeptidometric Assay for Enzymatically Active Prostate-Specific Antigen

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

Contact Info

Product

Resources

About