A novel type of bifunctional inhibitor directed against proteolytic activity and receptor/ligand interaction - Cystatin with a urokinase receptor binding site

Cancer invasion and metastasis is a process requiring a coordinated series of (anti-)adhesive, migratory, and pericellular proteolytic events involvin g various proteases such as urokinase-type plasminogen activator (uPA)/plas min, cathepsins B and L, and matrix metalloproteases. Novel types of double -headed inhibitors directed to different tumor-associated proteolytic syste ms were generated by substitution of a loop in chicken cystatin, which is n onessential for cysteine protease inhibition, with uPA-derived peptides cov ering the human uPA receptor binding sequence uPA-(19-31), The inhibition c onstants of these hybrids toward cysteine proteases are similar to those of wild-type cystatin (K-i, papain (pm), 1.9-2.4; K-i, cathepsin B (nM), 1.0- 1.7; K-i, cathepsin L (par), 0.12-0.61). FAGS analyses revealed that the hy brids compete for binding of uPA to the cell surface-associated uPA recepto r (uPAR) expressed on human U937 cells. The simultaneous interaction of the hybrid molecules with papain and uPAR was analyzed by surface plasmon reso nance. The measured K-D value of a papain-bound cystatin variant harboring the uPAR binding sequence of uPA (chCys-uPA-(19-31)) and soluble uPAR was 1 7 nM (K-D value for uPA/uPAR interaction, 5 nM). These results indicate tha t cystatins with a uPAR binding site are efficient inhibitors of cysteine p roteases and uPA/uPAR interaction at the same time. Therefore, these compac t and small bifunctional inhibitors may represent promising agents for the therapy of solid tumors.