| Popis: |
Cancer invasion and metastasis is a process requiring a coordinated series of (anti-)adhesive, migratory, and pericellular proteolytic events involving various proteases such as urokinase-type plasminogen activator (uPA)/plasmin, cathepsins B and L, and matrix metalloproteases. Novel types of double-headed inhibitors directed to different tumor-associated proteolytic systems were generated by substitution of a loop in chicken cystatin, which is nonessential for cysteine protease inhibition, with uPA-derived peptides covering the human uPA receptor binding sequence uPA-(19–31). The inhibition constants 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 (pm), 0.12–0.61). FACS analyses revealed that the hybrids compete for binding of uPA to the cell surface-associated uPA receptor (uPAR) expressed on human U937 cells. The simultaneous interaction of the hybrid molecules with papain and uPAR was analyzed by surface plasmon resonance. 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 17 nm (K D value for uPA/uPAR interaction, 5 nm). These results indicate that cystatins with a uPAR binding site are efficient inhibitors of cysteine proteases and uPA/uPAR interaction at the same time. Therefore, these compact and small bifunctional inhibitors may represent promising agents for the therapy of solid tumors. |
