Inhibition of the Interaction of Urokinase-Type Plasminogen Activator (uPA) with Its Receptor (uPAR) by Synthetic Peptides

Abstract

Focusing of the serine protease urokinase-type plasminogen activator (uPA) to the cell surface via interaction with its specific receptor (uPAR, CD87) is an important step for tumor cell invasion and metastasis. The ability of a synthetic peptide derived from the uPAR-binding region of uPA (comprising amino acids 16-32 of uPA; uPA(16-32)) to inhibit binding of fluorescently labeled uPA to uPAR on human promyeloid U937 cells was assessed by quantitative flow cytofluorometric analysis (FACS) and compared to the inhibitory capacities of other synthetic peptides known to interfere with uPA/uPAR-interaction. An about 3000-fold molar excess of uPA(16-32) resulted in 50% inhibition of pro-uPA binding to cell surface-associated uPAR. Using a solid-phase uPA-ligand binding assay employing recombinant soluble uPAR coated to microtiter plates, the minimal binding region of wild-type uPA was determined. The linear peptide uPA(19-31) and its more stable disulfide-bridged cyclic form (cyclo(19,31)uPA(19-31)) displayed uPAR-binding activity whereas other peptides such as uPA(18-30), uPA(20-32) or uPA(20-30) did not react with uPAR. Cyclic peptide derivatives of cyclo(19,31)uPA(19-31) in which certain amino acids were deleted and/or replaced by other amino acids as well as uPAR-derived wild-type peptides did also not inhibit uPA/uPAR-interaction. Therefore, the present investigations identified cyclo(19,31)uPA(19-31) as a potential lead structure for the development of uPA-peptide analogues to block uPA/uPAR-interaction.

Cloning and functional characterization of a new phosphatidyl-inositol anchored molecule of a metastasizing rat pancreatic tumor

“…Taking into account that there is ample evidence for the important of metalloproteinases and the plasminogen system in metastasis formation (Mareel et al, 1993;Stetler-Stevenons et al, 1993;Moller, 1993;DeClerck and Laug, 1996;Fazioli and Blasi, 1994;Andreasen et al, 1997;Testa and Quigley, 1990;Nagase, 1997;Crawford and Matrisian, 1995) and that much e ort is put into trials to prevent activation of these enzymes as a therapeutic regimen (Fazioli and Blasi, 1994;Burgle et al, 1997;Crowley et al, 1993), an interference with metastasis formation by blockade of the C4.4A molecule appears quite promising. Such an approach would also pro®t from the fact that expression of the C4.4A molecule in the adult is very restricted.…”

Role of plasminogen activator-plasmin system in tumor angiogenesis

“…Most approaches have focused on using linear or cyclic peptides based on the sequence of the growth factor domain of uPA [110,111]. Despite the promise of such peptides, their usefulness in vivo remains to be established.…”

Cyclo19,31[D-Cys19]-uPA19-31 Is a Potent Competitive Antagonist of the Interaction of Urokinase-Type Plasminogen Activator with Its Receptor (CD87)

“…The exchange of Cys19, Lys23, Tyr24, Phe25, Ile28, Trp30, and Cys31, respectively, by Ala resulted in peptides with strongly impaired uPAR-binding capacities, whereas replacement of the other amino acids had no or little effect on uPAR binding. Finally, the minimal uPAR-binding region of uPA was located to uPA 19-31 using synthetic peptides which were successively shortened from the amino-and/or carboxy-terminus starting with uPA 10-32 (Bürgle et al, 1997).…”

“…In uPA, Cys19 and Cys31, although in close proximity, form disulfide bonds with distinct cysteines (Cys11/Cys19 and Cys13/Cys31, respectively; Hansen et al, 1994a, b). The short distance between Cys19 and Cys31 in the native molecule may explain why peptide cyclo 19,31 uPA 19-31 with its 'illegitime' disulfide bond retains or displays an even increased uPAR-binding activity (Bürgle et al, 1997). Recently, we successfully generated double-headed inhibitors directed at different tumor-associated proteolytic systems by exchanging a disulfide-linked loop in cystatin, which is non-essential for cysteine protease inhibition, with uPA [19][20][21][22][23][24][25][26][27][28][29][30][31] .…”

“…All of the reactive peptides (with the clone 20 peptide being most active) harbor relatively short common motifs, such as LWXXY and FXXYLW, which may represent a variation of the motif 24 YFXXIXW 30 within the uPA molecule (only the critical hydrophobic amino acids for uPAR-binding are depicted) (Magdolen et al, 1996;Bürgle et al, 1997). Recently, a non-competitive, allosteric antagonist of prouPA/uPAR-interaction (Å6-peptide) derived from a non-receptor binding region of uPA ) has been identified (Guo et al, 2000).…”