Design of novel and selective inhibitors of urokinase-type plasminogen activator with improved pharmacokinetic properties for use as antimetastatic agents

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-Ser-Ser-4-amidinobenzylamide (inhibitor 26), inhibits uPA with a Ki 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 x 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.     

Discovery of GlyT1 inhibitors with improved pharmacokinetic properties

Glycine transporter 1 (GlyT1) represents a novel target for the treatment of schizophrenia via the potentiation of glutamatergic NMDA receptors. The discovery of 4,4-disubstituted piperidine inhibitors of GlyT1 which exhibit improved pharmacokinetic properties, including oral bioavailability, is discussed.     

Gonadotropin surge-induced up-regulation of the plasminogen activators (tissue plasminogen activator and urokinase plasminogen activator) and the urokinase plasminogen activator receptor within bovine periovulatory follicular and luteal tissue

This study examined the effect of the preovulatory gonadotropin surge on the temporal and spatial regulation of tissue plasminogen activator (tPA), urokinase plasminogen activator (uPA), and uPA receptor (uPAR) mRNA expression and tPA, uPA, and plasmin activity in bovine preovulatory follicles and new corpora lutea collected at approximately 0, 6, 12, 18, 24, and 48 h after a GnRH-induced gonadotropin surge. Messenger RNAs for tPA, uPA, and uPAR were increased in a temporally specific fashion within 24 h of the gonadotropin surge. Localization of tPA mRNA was primarily to the granulosal layer, whereas both uPA and uPAR mRNAs were detected in both the granulosal and thecal layers and adjacent ovarian stroma. Activity for tPA was increased in follicular fluid and the preovulatory follicle apex and base within 12 h after the gonadotropin surge. The increase in tPA activity in the follicle base was transient, whereas the increased activity in the apex was maintained through the 24 h time point. Activity for uPA increased in the follicle apex and base within 12 h of the gonadotropin surge and remained elevated. Plasmin activity in follicular fluid also increased within 12 h after the preovulatory gonadotropin surge and was greatest at 24 h. Our results indicate that mRNA expression and enzyme activity for both tPA and uPA are increased in a temporally and spatially specific manner in bovine preovulatory follicles after exposure to a gonadotropin surge. Increased plasminogen activator and plasmin activity may be a contributing factor in the mechanisms of follicular rupture in cattle.     

Crystals of urokinase type plasminogen activator complexes reveal the binding mode of peptidomimetic inhibitors

Urokinase type plasminogen activator (uPA), a trypsin-like serine proteinase, plays an important role in normal tissue re-modelling, cell adhesion, and cell motility. In addition, studies utilizing normal animals and potent, selective uPA inhibitors or genetically modified mice that lack functional uPA genes have demonstrated that uPA can significantly enhance tumor initiation, growth, progression and metastasis, strongly suggesting that this enzyme may be a promising anti-cancer target. We have investigated the structure-activity relationship (SAR) of peptidomimetic inhibitors of uPA and solved high resolution X-ray structures of key, lead small molecule inhibitors (e.g. phenethylsulfonamidino(P4)-D-seryl(P3)-L-alanyl(P2)-L-argininal(P1) and derivatives thereof) in complex with the uPA proteinase domain. These potent inhibitors are highly selective for uPA. The non-natural D-seryl residue present at the P3 position in these inhibitors contributes substantially to both potency and selectivity because, due to its D-configuration, its side-chain binds in the S4 pocket to interact with the uPA unique residues Leu97b and His99. Additional potency and selectivity can be achieved by optimizing the inhibitor P4 residue to bind a pocket, known as S1sub or S1beta, that is adjacent to the primary specificity pocket of uPA.     

Antigen levels of urokinase type plasminogen activator and its inhibitors in primary breast cancer

Primary Breast Cancer, Urokinase-Type Plasminogen Activator, Inhibitors The aim of the study was to monitor urokinase plasminogen activator antigen concentrations and its type 1 (PAI-1) and type 2 (PAI-2) inhibitors in histologically defined forms of primary breast cancer and a comparison with these antigens levels in normal tissue. Another goal was a search for a relationship/or its lack/between the occurrence of the new generation markers of neoplastic disease and a presence/or absence/of lymph node metastases. U-PA, PAI-1 and PAI-2 antigen levels were determined by ELISA tests in protein extracts of breast cancer tissues. Among the studied breast tumors 32 specimens were ductal carcinomas, 15 specimens were lobular carcinomas and the remaining 13 were other rare histological forms. In comparison to the obtained values of u-PA antigen levels in normal tissue, the values in neoplastic tissues were elevated several times: 11-fold, 6-fold and 15-fold in ductal c., lobular c. and other rare neoplasms. The values of PAI-1 antigen levels were about 20-fold higher for all studied, histologically defined primary breast cancers. The greatest differences of PAI-2 antigen levels growth was observed in histologically defined primary breast cancer forms. It was augmented 10-fold, 40-fold and 20-fold, respectively, for ductal carcinoma, lobular carcinoma and rare forms of neoplasms. In various forms of invasive breast cancer and those without lymph node metastases the content of u-PA, PAI-1 and PAI-2 were also significantly elevated. Among the new generation of independent markers of the neoplastic process, PAI-2 seems to be the most reliable marker for the identification of primary breast cancer. The goal of the present study was to evaluate a possible combined prognostic value of the three major components of the u-PA system (u-PA, PAI-1 and PAI-2) in patients with defined histopathological forms of primary breast cancer.     

Intermolecular contact regions in urokinase plasminogen activator receptor

The glycolipid-anchored urokinase-type plasminogen activator receptor (uPAR) is engaged in various signal transduction events related to cell adhesion, migration and proliferation. In this study, using phage display and peptide array techniques, we have identified several intermolecular contact regions of uPAR. Phage-displayed uPAR fragments bound to immobilized soluble uPAR on magnetic beads, revealing that regions uPAR-(7-28) and uPAR-(60-91) in domain I, uPAR-(101-121) in domain II and uPAR-(240-260) in domain III are possible uPAR-uPAR contact sites. Using peptide array, two additional sites could be identified, uPAR-(51-59) in domain I and uPAR-(144-155) in domain II. The putative uPAR-uPAR interaction sites are different from the previously identified uPA-binding sites. Functionally, peptides uPAR-(84-95) and uPAR-(240-248) could partially inhibit differentiated human U937 monocyte adhesion to vitronectin in the presence of uPA, indicating that these two uPAR regions might be involved not only in uPAR-uPAR but also in uPAR-vitronectin interactions. We propose that multiple uPAR-uPAR ectodomain interactions contribute considerably to the regulation of various cellular functions of uPAR.     

Tissue plasminogen activator and urokinase enhance the binding of plasminogen to thrombospondin

Thrombospondin (TSP) is a multifunctional platelet alpha-granule and extracellular matrix glycoprotein that binds specifically to plasminogen (Plg) via that protein's lysine-binding site and modulates activation by tissue activator (TPA). In this study we report that the plasminogen activators, TPA and urokinase, greatly influence the binding of Plg to TSP. Using an enzyme-linked immunosorbent assay and a TSP-Sepharose affinity bead-binding assay we have found that Plg-TSP complex formation was markedly enhanced (up to 5-fold) when catalytic concentrations of Plg activators were included in the reaction mixtures. The enhancement was dependent upon the generation of small amounts of active plasmin and was duplicated by pretreatment of the immobilized TSP with plasmin prior to addition of the Plg. The enhancement effect was associated with selective proteolysis of the immobilized TSP. Purified Lys-Plg (the plasmin modified form of native Glu-Plg) bound to TSP to a greater extent than Glu-Plg, and binding of both forms was augmented by Plg activators. The apparent KD values of complex formation were unchanged in the presence of Plg activators suggesting that the enhancement effect was due to the generation of additional binding sites. The increased amount of bound Plg was demonstrated to result in a similar increase in the amount of plasmin generated from the complexes by TPA. Plg activators did not influence binding of Plg to histidine-rich glycoprotein or of histidine-rich glycoprotein to TSP, demonstrating specificity. In addition when TSP was treated with other proteases (human thrombin or human leukocyte elastase) no augmentation of Plg binding was seen. Thus, the initial production of small amounts of plasmin from Plg immobilized on TSP in fibrin-free microenvironments could generate a positive feedback loop by enzymatically modifying both TSP and Plg, resulting in an increase in TSP-Plg complex formation leading to the localized production of substantially more plasmin.     

Decreased plasminogen activator inhibitor-1 secretion in hypoxic corneal epithelial cells is associated with increased urokinase plasminogen activator activity

The aim of this study was to determine the effects of hypoxia on mRNA levels, cell-associated and -secreted protein concentration, activity, and protein complex formation of urokinase-type plasminogen activator, its receptor, and plasminogen activator inhibitor type-1 in corneal epithelium. Non-transformed human corneal epithelial cells were cultured in 20% oxygen (normoxic conditions) or 2% oxygen (hypoxic conditions) for 1, 3, 5, or 7 days. Relative changes in mRNA levels of plasminogen activator, receptor, and plasminogen activator inhibitor-1 were determined using a cDNA expression array, chemiluminescence, and densitometry. Protein concentrations were determined using enzyme linked immunosorbent assays. Activity assays were also used. Protein complex formation was assayed using cell surface biotinylation, immunoprecipitation, and Western blot analysis. Hypoxic corneal epithelial cells demonstrated no significant differences in plasminogen activator or receptor mRNA. Cell-associated plasminogen activator and membrane-associated receptor protein levels were unchanged. In contrast decreases in mRNA and secreted plasminogen activator inhibitor-1 protein were observed in hypoxic cells. Concurrently, increased cell-associated plasminogen activator activity was observed in hypoxic cells. The formation of plasminogen activator/receptor/plasminogen activator inhibitor-1 complex at the cell surface was not inhibited by hypoxia. However, in hypoxic cells less plasminogen activator inhibitor-1 was associated with receptor. It is concluded that in corneal epithelium cultured in 2% oxygen plasminogen activator inhibitor-1 may be an important regulatory factor of the plasminogen activator system resulting in increased urokinase plasminogen activator activity.     

Mechanisms of cardiac fibrosis induced by urokinase plasminogen activator

Human hearts with end-stage failure and fibrosis have macrophage accumulation and elevated plasminogen activator activity. However, the mechanisms that link macrophage accumulation and plasminogen activator activity with cardiac fibrosis are unclear. We previously reported that mice with macrophage-targeted overexpression of urokinase plasminogen activator (SR-uPA+/o mice) develop cardiac macrophage accumulation by 5 weeks of age and cardiac fibrosis by 15 weeks. We used SR-uPA+/o mice to investigate mechanisms through which macrophage-expressed uPA causes cardiac macrophage accumulation and fibrosis. We hypothesized that: 1) macrophage accumulation and cardiac fibrosis in SR-uPA+/o mice are dependent on localization of uPA by the uPA receptor (uPAR); 2) activation of plasminogen by uPA and subsequent activation of transforming growth factor-beta1 (TGF-beta1) and matrix metalloproteinase (MMP)-2 and -9 by plasmin are critical pathways through which uPA-expressing macrophages accumulate in the heart and cause fibrosis; and 3) uPA-induced cardiac fibrosis can be attenuated by treatment with verapamil. To test these hypotheses, we bred the SR-uPA+/o transgene into mice deficient in either uPAR or plasminogen and measured cardiac macrophage accumulation and fibrosis. We also measured cardiac TGF-beta1 protein (total and active), Smad2 phosphorylation, and MMP activity after the onset of macrophage accumulation but before the onset of cardiac fibrosis. Finally, we treated mice with verapamil. Our studies revealed that plasminogen is necessary for uPA-induced cardiac fibrosis and macrophage accumulation but uPAR is not. We did not detect plasmin-mediated activation of TGF-beta1, MMP-2, or MMP-9 in hearts of SR-uPA+/o mice. However, verapamil treatment significantly attenuated both cardiac fibrosis and macrophage accumulation.     

Structure-based design, synthesis and SAR of a novel series of thiopheneamidine urokinase plasminogen activator inhibitors

The serine protease urokinase plasminogen activator (uPA) is thought to play a central role in tumor metastasis and angiogenesis. Molecular modeling studies suggest that 5-thiomethylthiopheneamidine inhibits uPA by binding at the S1 pocket of the active site. Further structure based elaboration of this residue resulted in a novel class of potent and selective inhibitors of uPA.     

Characterization of urokinase type plasminogen activator modified by phenylglyoxal

A chemical modification of single-chain urokinase-type plasminogen activator (scu-PA) with phenylglyoxal under mild conditions resulted in the scu-PA derivatives with various numbers of the modified Arg residues. The study of properties of the resulting derivatives demonstrated that the modification of 4-12 Arg residues did not cause any loss of the activator, fibrinolytic, and potential amidase activities of the activator. The scu-PA with four modified Arg residues was found to be the most stable derivative in human blood plasma; it causes a more efficient lysis of plasma clots than the native activator. Three of four modified Arg residues are supposed to be within the 178RRHRGGS184 cluster, which was localized in the superficial loop of the scu-PA globule and was shown to interact with the complementary series of negatively charged residues in the molecule of the main plasma inhibitor PAI-1. The neutralization of positively charged Arg residues in this cluster decreases the affinity of scu-PA and the double chain urokinase-type plasminogen activator for PAI-1, which results in an enhancement of the stability in plasma and the fibrinolytic efficiency of the activator. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2002, vol. 28, no. 4; see also http://www.maik.ru.     

Kinetic analysis of the interactions between plasminogen activator inhibitor 1 and both urokinase and tissue plasminogen activator

Plasminogen activator inhibitor 1 (PAI-1) was purified from medium conditioned by cultured bovine aortic endothelial cells by successive chromatography on concanavalin A Sepharose, Sephacryl S-200, Blue B agarose, and Bio-Gel P-60. As shown previously for conditioned media (C. M. Hekman and D. J. Loskutoff (1985) J. Biol. Chem. 260, 11581-11587) the purified PAI-1 preparation contained latent inhibitory activity which could be stimulated 9.4-fold by sodium dodecyl sulfate and 45-fold by guanidine-HCl. The specific activity of the preparation following treatment with 0.1% sodium dodecyl sulfate was 2.5 X 10(3) IU/mg. The reaction between purified, guanidine-activated PAI-1 and both urokinase and tissue plasminogen activator (tPA) was studied. The second-order rate constants (pH 7.2, 35 degrees C) for the interaction between guanidine-activated PAI-1 and urokinase (UK), and one- and two-chain tPA are 1.6 X 10(8), 4.0 X 10(7), and 1.5 X 10(8) M-1 S-1, respectively. The presence of CNBr fibrinogen fragments had no affect on the rate constants of either one- or two-chain tPA. Steady-state kinetic analysis of the effect of PAI-1 on the rate of plasminogen activation revealed that the initial UK/PAI-1 interaction can be competed with plasminogen suggesting that the UK/PAI-1 interaction may involve a competitive type of inhibition. In contrast, the initial tPA/PAI-1 interaction can be competed only partially with plasminogen, suggesting that the tPA/PAI-1 interaction may involve a mixed type of inhibition. The results indicate that PAI-1 interacts more rapidly with UK and tPA than any PAI reported to date and suggest that PAI-1 is the primary physiological inhibitor of single-chain tPA. Moreover, the interaction of PAI-1 with tPA differs from its interaction with UK, and may involve two sites on the tPA molecule.     

PEGylated leuprolide with improved pharmacokinetic properties

Leuprolide, a gonadotropin-releasing hormone (GnRH) agonist widely used in androgen deprivation therapy for the treatment of advanced prostate cancer, suffers from a short circulating half-life like other peptide therapeutics. As an attempt to improve its pharmacokinetic properties, two PEGylated leuprolides with different molecular weight were synthesized utilizing N-hydroxysuccinimidyl (NHS) conjugation chemistry. The reaction conditions, including reaction temperature, reaction time and feed ratio of the reactants, were optimized to obtain a higher yield. Reverse-phase high performance liquid chromatography (RP-HPLC) characterization indicates a high purity of the resulting conjugates. Matrix-assisted laser desorption mass spectrometry (MALDI-MS) characterization suggests a 1:1 PEGylation. ¹H NMR study reveals that the reaction occurs on the imidazolyl group on the histidine residue and the conjugates are stable in pH7.4 aqueous solutions. The in vitro bioactivity of the conjugates was evaluated using both hormone-sensitive and hormone-insensitive cell lines. It was found that the PEGylated peptides can still counteract the stimulatory action of androgens and the mitogenic action of epidermal growth factor on cell proliferation. The in vivo bioactivity of the conjugates was also tested. Like the unmodified peptide, administration of the conjugates to male rats leads to an initial testosterone surge, followed by a suppression of testosterone secretion. Pharmacokinetics of the drugs after i.v. and s.c. administrations were determined. In both cases, a prolonged circulating half-life, an increased AUC, and a decreased Cl_F were observed for the PEGylated drugs.     

Discovery of selective urokinase plasminogen activator (uPA) inhibitors as a potential treatment for multiple sclerosis

We report here the design and synthesis of a novel series of benzylamines that are potent and selective inhibitors of uPA with promising oral availability in rat. Further evaluation of one representative (ZK824859) of the new structural class showed that this compound lowered clinical scores when dosed in either acute or chronic mouse EAE models, suggesting that uPA inhibitors of this type could be useful for the treatment of multiple sclerosis.     

RNA interference-directed knockdown of urokinase plasminogen activator and urokinase plasminogen activator receptor inhibits prostate cancer cell invasion, survival, and tumorigenicity in vivo

The invasive ability of tumor cells plays a key role in prostate cancer metastasis and is a major cause of treatment failure. Urokinase plasminogen activator-(uPA) and its receptor (uPAR)-mediated signaling have been implicated in tumor cell invasion, survival, and metastasis in a variety of cancers. This study was undertaken to investigate the biological roles of uPA and uPAR in prostate cancer cell invasion and survival, and the potential of uPA and uPAR as targets for prostate cancer therapy. uPA and uPAR expression correlates with the metastatic potential of prostate cancer cells. Thus, therapies designed to inhibit uPA and uPAR expression would be beneficial. LNCaP, DU145, and PC3 are prostate cancer cell lines with low, moderate, and high metastatic potential, respectively, as demonstrated by their capacity to invade the extracellular matrix. In this study we utilized small hairpin RNAs (shRNAs), also referred to as small interfering RNAs, to target human uPA and uPAR. These small interfering RNA constructs significantly inhibited uPA and uPAR expression at both the mRNA and protein levels in the highly metastatic prostate cancer cell line PC3. Our data demonstrated that uPA-uPAR knockdown in PC3 cells resulted in a dramatic reduction of tumor cell invasion as indicated by a Matrigel invasion assay. Furthermore, simultaneous silencing of the genes for uPA and uPAR using a single plasmid construct expressing shRNAs for both uPA and uPAR significantly reduced cell viability and ultimately resulted in the induction of apoptotic cell death. RNA interference for uPA and uPAR also abrogated uPA-uPAR signaling to downstream target molecules such as ERK1/2 and Stat 3. In addition, our results demonstrated that intratumoral injection with the plasmid construct expressing shRNAs for uPA and uPAR almost completely inhibited established tumor growth and survival in an orthotopic mouse prostate cancer model. These findings uncovered evidence of a complex signaling network operating downstream of uPA-uPAR that actively advances tumor cell invasion, proliferation, and survival of prostate cancer cells. Thus, RNA interference-directed targeting of uPA and uPAR is a convenient and novel tool for studying the biological role of the uPA-uPAR system and raises the potential of its application for prostate cancer therapy.     

Secretion of macrophage urokinase plasminogen activator is dependent on proteoglycans.

The importance of proteoglycans for secretion of proteolytic enzymes was studied in the murine macrophage cell line J774. Untreated or 4beta-phorbol 12-myristate 13-acetate (PMA)-stimulated macrophages were treated with hexyl-beta-d-thioxyloside to interfere with the attachment of glycosaminoglycan chains to their respective protein cores. Activation of the J774 macrophages with PMA resulted in increased secretion of trypsin-like serine proteinase activity. This activity was completely inhibited by plasminogen activator inhibitor 1 and by amiloride, identifying the activity as urokinase plasminogen activator (uPA). Treatment of both the unstimulated or PMA-stimulated macrophages with xyloside resulted in decreased uPA activity and Western blotting analysis revealed an almost complete absence of secreted uPA protein after xyloside treatment of either control- or PMA-treated cells. Zymography analyses with gels containing both gelatin and plasminogen confirmed these findings. The xyloside treatment did not reduce the mRNA levels for uPA, indicating that the effect was at the post-translational level. Treatment of the macrophages with xylosides did also reduce the levels of secreted matrix metalloproteinase 9. Taken together, these findings indicate a role for proteoglycans in the secretion of uPA and MMP-9.