Design of Novel and Selective Inhibitors of Urokinase-type Plasminogen Activator with Improved Pharmacokinetic Properties for Use as Antimetastatic Agents

Abstract

The serine protease urokinase-type plasminogen activator (uPA) interacts with a specific receptor (uPAR) on the surface of various cell types, including tumor cells, and plays a crucial role in pericellular proteolysis. High levels of uPA and uPAR often correlate with poor prognosis of cancer patients. Therefore, the specific inhibition of uPA with small molecule active-site inhibitors is one strategy to decrease the invasive and metastatic activity of tumor cells. We have developed a series of highly potent and selective uPA inhibitors with a C-terminal 4-amidinobenzylamide residue. Optimization was directed toward reducing the fast elimination from circulation that was observed with initial analogues. The x-ray structures of three inhibitor/uPA complexes have been solved and were used to improve the inhibition efficacy. One of the most potent and selective derivatives, benzylsulfonyl-D-SerSer-4-amidinobenzylamide (inhibitor 26), inhibits uPA with a K i of 20 nM. This inhibitor was used in a fibrosarcoma model in nude mice using lacZ-tagged human HT1080 cells, to prevent experimental lung metastasis formation. Compared with control (100%), an inhibitor dose of 2 ؋ 1.5 mg/kg/day reduced the number of experimental metastases to 4.6 ؎ 1%. Under these conditions inhibitor 26 also significantly prolonged survival. All mice from the control group died within 43 days after tumor cell inoculation, whereas 50% of mice from the inhibitor-treated group survived more than 117 days. This study demonstrates that the specific inhibition of uPA by these inhibitors may be a useful strategy for the treatment of cancer to prevent metastasis.The detachment of malignant cells from the primary tumor and their subsequent migration within the surrounding tissue, including intravasation and extravasation into blood and lymph vessels, leads to tumor dissemination and the formation of metastases at distant loci. Whereas a solid tumor can be removed by surgery or treated by radio-, chemo-, or hormone therapy, invasive tumor cells that have spread over the whole body can form secondary tumors leading to poor prognosis or death of cancer patients. Several proteases, such as matrix metalloproteases, the cysteine proteases cathepsin B and L, the aspartyl protease cathepsin D, and serine proteases, e.g. plasmin and uPA, 1 are involved at multiple stages during growth, invasion and progression of tumors, including metastases formation (1). High levels of expression of these proteases often correlate with poor prognosis for cancer patients (2). However, in several clinical cancer trials with different types of nonspecific matrix metalloprotease inhibitors, disappointing results with poor benefit and severe side effects were observed (3). This stimulated the search for alternative proteases with matrix degrading activity as new targets for anti-cancer drugs. An important role in metastasis has been recently ascribed to the plasmin-plasminogen activation system and especially to uPA.Both, uPA and the second endogenous plasminogen activator tPA are...

RNA Interference-directed Knockdown of Urokinase Plasminogen Activator and Urokinase Plasminogen Activator Receptor Inhibits Prostate Cancer Cell Invasion, Survival, and Tumorigenicity in Vivo

“…UPA-or uPAR-neutralizing antibodies (33), protease inhibitors (34), urokinase-specific inhibitors (5,35,36), uPA or uPAR antisense oligonucleotides (11,16,37,38), and small molecules that target uPA or uPAR (39 40) are being investigated as possible strategies to block either uPA or uPAR activity, thereby causing the suppression of tumor growth, invasion, and metastasis.…”

The role of Med19 in the proliferation and tumorigenesis of human hepatocellular carcinoma cells

“…Recently emerging as an important tool for silencing specific genes, RNAi is more potent in inhibiting gene expression than other methods and carries less risk of toxicity [21][22][23] . More importantly, with the effectiveness and relative ease with which dsRNA can be introduced into cells and tissues, RNAi has a significant therapeutic potential for targeting human carcinomas.…”

Geldanamycins exquisitely inhibit HGF/SF-mediated tumor cell invasion

“…When Met signaling is initiated by HGF/SF, both uPA and uPAR expression are upregulated and plasminogen is consequently cleaved into plasmin, a sequence of events that results in degradation of the extracellular matrix (Ellis et al, 1993). High levels of uPA and uPAR expression are associated with poor clinical prognosis (Duffy, 1996;Duffy et al, 1996;Harbeck et al, 2002), and uPAR-targeted anticancer strategies are being developed Lakka et al, 2003;Schweinitz et al, 2004). Our laboratory previously developed a cell-based method for screening HGF/SF-induced uPA-plasmin network inhibitors and, by using this assay, discovered that ).…”