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Abstract
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.
Highlights
Cancer invasion and metastasis is a process requiring a coordinated series ofadhesive, migratory, and pericellular proteolytic events involving various proteases such as urokinase-type plasminogen activator/plasmin, cathepsins B and L, and matrix metalloproteases
Design, Cloning, Expression, and Purification of Cystatin/urokinase-type plasminogen activator (uPA) Variants—Cystatins, the natural inhibitors of cysteine proteases, as well as uPA-derived peptides, which abrogate uPA/uPA receptor (uPAR) interaction are well known to reduce the invasive capacity of tumor cells in vitro and in vivo
The loop regions located between the disulfide-bridged Cys71/Cys81 in chicken cystatin and Cys73/Cys83 in that of human cystatin C were substituted by uPA-(19 –31) and uPA-(21–30)[S21C, H29C], respectively (Fig. 1)
Summary
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 measured KD value of a papain-bound cystatin variant harboring the uPAR binding sequence of uPA (chCys-uPA-(19 –31)) and soluble uPAR was 17 nM (KD 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.
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