Selective urokinase-type plasminogen activator (uPA) inhibitors. Part 1: 2-Pyridinylguanidines.

The identification of 2-pyridinylguanidines (e.g., 27 and 28) as selective inhibitors of urokinase-type plasminogen activator (uPA) is described. The X-ray crystal structure of 27 has been determined, and modelling has been used to predict binding in the enzyme active site.     

Selective urokinase-type plasminogen activator (uPA) inhibitors. Part 2: (3-Substituted-5-halo-2-pyridinyl)guanidines.

Based on previous modeling predictions, a series of (3-substituted-5-chloro-2-pyridinyl)guanidines have been designed with good potency and selectivity for urokinase-type plasminogen activator (uPA). Compound 36 has a K(i) of 0.17 microM and greater than 300-fold selectivity with respect to tPA and plasmin.     

Synthesis and evaluation of non-basic inhibitors of urokinase-type plasminogen activator (uPA)

Recent drug discovery programs targeting urokinase plasminogen activator (uPA) have resulted in nonpeptidic inhibitors consisting of amidine or guanidine functional groups attached to aromatic or heteroaromatic scaffolds. There is a general problem of poor oral bioavailability of these charged inhibitors. In this paper, we report the synthesis and evaluation of a series of naphthamide and naphthalene sulfonamides as uPA inhibitors containing non-basic groups as substitute for amidine or guanidine groups.     

Synthesis and preliminary evaluation of amiloride analogs as inhibitors of the urokinase-type plasminogen activator (uPA)

A known side-activity of the oral potassium-sparing diuretic drug amiloride is inhibition of the enzyme urokinase-type plasminogen activator (uPA, K(i)=7 μM), a promising anticancer target. Several studies have demonstrated significant antitumor/metastasis properties for amiloride in animal cancer models and it would appear that these arise, at least in part, through inhibition of uPA. Selective optimization of amiloride's structure for more potent inhibition of uPA and loss of diuretic effects would thus appear as an attractive strategy towards novel anticancer agents. The following report is a preliminary structure-activity exploration of amiloride analogs as inhibitors of uPA. A key finding was that the well-studied 5-substituted analogs ethylisopropyl amiloride (EIPA) and hexamethylene amiloride (HMA) are approximately twofold more potent than amiloride as uPA inhibitors.     

Prognostic and predictive value of the urokinase-type plasminogen activator (uPA) and its inhibitors PAI-1 and PAI-2 in operable breast cancer

The present study on the prognostic and predictive value of serine proteases was conducted in 460 early breast cancer patients mostly treated with some kind of adjuvant systemic therapy: 156 received chemotherapy, 141 hormone therapy and 111 a combination of both. Already in univariate analysis PAI-1 was the only proteolytic factor with a significant impact on DFS, which was retained in multivariate analysis (p = 0.020); PAI-2 showed borderline significance in univariate analysis (p = 0.0503) and uPA did not present as a significant prognostic factor for DFS in our patient series. In a separate univariate analysis of DFS on patient subgroups defined by adjuvant systemic therapy, a higher risk of relapse associated with higher uPA and PAI-1 levels was found in the subgroup of patients who did not receive any treatment; this difference did not reach the level of significance, probably due to the small number (n = 52) of patients in this group (HR 1.37; p = 0.71 and HR 2.14; p = 0.321, respectively). A higher risk of relapse was also found in the subgroup of patients treated with adjuvant chemotherapy (HR 1.44; p = 0.381 and HR 2.48; p = 0.003, respectively). In contrast, the bad prognostic impact of high uPA and PAI-1 levels was lost in the subgroup of patients treated with adjuvant hormone therapy (HR 0.79; p = 0.693 and HR 0.26; p = 0.204, respectively). The same observations were made for the uPA/PAI-1 combination. Our study confirmed the prognostic value of serine proteases in early breast cancer. In addition, it pointed to a possible predictive value of these tumor markers for response to adjuvant hormone therapy with tamoxifen, which should be confirmed in further studies.     

Renal urokinase-type plasminogen activator (uPA) receptor but not uPA deficiency strongly attenuates ischemia reperfusion injury and acute kidney allograft rejection

Central mechanisms leading to ischemia induced allograft rejection are apoptosis and inflammation, processes highly regulated by the urokinase-type plasminogen activator (uPA) and its specific receptor (uPAR). Recently, up-regulation of uPA and uPAR has been shown to correlate with allograft rejection in human biopsies. However, the causal connection of uPA/uPAR in mediating transplant rejection and underlying molecular mechanisms remain poorly understood. In this study, we evaluated the role of uPA/uPAR in a mice model for kidney ischemia reperfusion (IR) injury and for acute kidney allograft rejection. uPAR but not uPA deficiency protected from IR injury. In the allogenic kidney transplant model, uPAR but not uPA deficiency of the allograft caused superior recipient survival and strongly attenuated loss of renal function. uPAR-deficient allografts showed reduced generation of reactive oxygen species and apoptosis. Moreover, neutrophil and monocyte/macrophage infiltration was strongly attenuated and up-regulation of the adhesion molecule ICAM-1 was completely abrogated in uPAR-deficient allografts. Inadequate ICAM-1 up-regulation in uPAR(-/-) primary aortic endothelial cells after C5a and TNF-alpha stimulation was confirmed by in vitro experiments. Our results demonstrate that the local renal uPAR plays an important role in the apoptotic and inflammatory responses mediating IR-injury and transplant rejection.     

Structural basis for recognition of urokinase-type plasminogen activator by plasminogen activator inhibitor-1

Plasminogen activator inhibitor-1 (PAI-1), together with its physiological target urokinase-type plasminogen activator (uPA), plays a pivotal role in fibrinolysis, cell migration, and tissue remodeling and is currently recognized as being among the most extensively validated biological prognostic factors in several cancer types. PAI-1 specifically and rapidly inhibits uPA and tissue-type PA (tPA). Despite extensive structural/functional studies on these two reactions, the underlying structural mechanism has remained unknown due to the technical difficulties of obtaining the relevant structures. Here, we report a strategy to generate a PAI-1·uPA(S195A) Michaelis complex and present its crystal structure at 2.3-Å resolution. In this structure, the PAI-1 reactive center loop serves as a bait to attract uPA onto the top of the PAI-1 molecule. The P4–P3′ residues of the reactive center loop interact extensively with the uPA catalytic site, accounting for about two-thirds of the total contact area. Besides the active site, almost all uPA exosite loops, including the 37-, 60-, 97-, 147-, and 217-loops, are involved in the interaction with PAI-1. The uPA 37-loop makes an extensive interaction with PAI-1 β-sheet B, and the 147-loop directly contacts PAI-1 β-sheet C. Both loops are important for initial Michaelis complex formation. This study lays down a foundation for understanding the specificity of PAI-1 for uPA and tPA and provides a structural basis for further functional studies.     

Antigen-driven lymphocyte recruitment to the lung is diminished in the absence of urokinase-type plasminogen activator (uPA) receptor, but is independent of uPA.

The requirement for urokinase plasminogen activator (uPA) and uPA receptor (uPAR) in T lymphocyte migration is unknown. uPA(-/-) mice have fewer pulmonary lymphocytes in response to certain infections, but its unknown whether this is due to diminished recruitment. Primed, recipient mice were IT inoculated with Ag. Three days later, fluorescently labeled lymphoblasts from background-matched control wild-type (WT), uPA(-/-), or uPAR(-/-) donor mice were injected i.v., and their recruitment was determined. Approximately twice the number of uPA(-/-) compared with WT lymphoblasts were recruited to the lungs of WT recipients. This difference was eliminated when uPA(-/-) and WT lymphoblasts were injected into uPA(-/-) recipients. Thus, the reduced number of lung lymphocytes in infected uPA(-/-) mice is not due to reduced recruitment. However, uPAR is critically involved in recruitment. Markedly fewer uPAR(-/-) compared with WT lymphoblasts were recruited to the lung. These findings suggest that uPAR may be a novel target for immune modulation in T lymphocyte-mediated disorders.     

2-Amidino analogs of glycine-amiloride conjugates: inhibitors of urokinase-type plasminogen activator

The relative non-toxicity of the diuretic amiloride, coupled with its selective inhibition of the protease urokinase plasminogen activator (uPA), makes this compound class attractive for structure-activity studies. Herein we substituted the C(2)-acylguanidine of C(5)-glycyl-amiloride with amidine and amidoxime groups. The data show the importance of maintaining C(5)-hydrophobicity. The C(5)-benzylglycine analogs containing either C(2)-acylguanidine or amidine inhibited uPA with an IC(50) ranging from 3 to 7 μM and were cytotoxic to human U87 malignant glioma cells.     

2-(2-Hydroxy-3-alkoxyphenyl)-1H-benzimidazole-5-carboxamidine derivatives as potent and selective urokinase-type plasminogen activator inhibitors

The development of potent and selective urokinase-type plasminogen activator (uPA) inhibitors based on the lead molecule 2-(2-hydroxy-3-ethoxyphenyl)-1H-benzimidazole-5-carboxamidine (3a) is described.     

Inhibition of NF-kappa B-Rel A expression by antisense oligodeoxynucleotides suppresses synthesis of urokinase-type plasminogen activator (uPA) but not its inhibitor PAI-1.

The essential role of urokinase-type plasminogen activator (uPA) in tumor invasion and metastasis stresses the necessity of a fine-tuned cellular control over its expression. It has been shown that changes in uPA directly correlate with changes in cell invasiveness. We examined the role of Rel-related proteins in uPA synthesis by human ovarian cancer cells by inhibiting their expression using the antisense (AS) oligodeoxynucleotide (ODN) technology. Exposure of OV-MZ-6 cells to 10 microM phosphorothioate (PS)-derivatized AS-ODN directed to Rel A led to a maximal 50% decrease of uPA antigen in cell lysates and a 70% reduction in cell cultures supernatants accompanied by a significant transient decline in uPA mRNA levels. Antisense-PS-ODN directed to NF-kappa B1 (p50) or c-rel had no effect on uPA protein expression. AS-PS-ODN directed to Rel A also affected the proteolytic capacity of OV-MZ-6 cells reflected by an approximately 70% decrease in the fibrinolytic capacity of the cells within 24 h compared to untreated controls. AS-PS-ODN directed to I kappa B alpha expression increased uPA in cell culture supernatants up to 50%. uPA receptor (uPAR) production and synthesis of plasminogen activator inhibitor type-1 (PAI-1) were not altered by either AS-PS-ODN applied. Western blot and gel retardation analyses revealed constitutive expression of Rel-related proteins in nuclear protein extracts of OV-MZ-6 cells. Thus these proteins seem to be implicated in uPA regulation and may thereby contribute to tumor spread and metastasis.     

Plasminogen activator inhibitor-1 (PAI-1) and urokinase plasminogen activator (uPA) in sputum of allergic asthma patients

Urokinase plasminogen activator (uPA) and its inhibitor (PAI-1) have been associated with asthma. The aim of this study was to evaluate concentration of uPA and PAI-1 in induced sputum of house dust mite allergic asthmatics (HDM-AAs). The study was performed on 19 HDM-AAs and 8 healthy nonatopic controls (HCs). Concentration of uPA and PAI-1 was evaluated in induced sputum supernatants using ELISA method. In HDM-AAs the median sputum concentration of uPA (128 pg/ml; 95% CI 99 to 183 pg/ml) and PAI-1 (4063 pg/ml; 95%CI 3319 to 4784 pg/ml) were significantly greater than in HCs (17 pg/ml; 95%CI 12 to 32 pg/ml; p<0.001 and 626 pg/ml; 95%CI 357 to 961 pg/ml; p<0.001 for uPA and PAI-1 respectively). The sputum concentration of uPA correlated with sputum total cell count (r=0.781; p=0.0001) and with logarithmically transformed exhaled nitric oxide concentration (eNO) (r=0.486; p=0.035) but not with FEV1 or bronchial reactivity to histamine. On the contrary, the sputum PAI-1 concentration correlated with FEV1 (r=-0,718; p=0.0005) and bronchial reactivity to histamine expressed as log(PC20) (r=-0.824; p<0.0001) but did not correlate with sputum total cell count or eNO. The results of this study support previous observations linking PAI-1 with airway remodeling and uPA with cellular inflammation. Moreover, the observed effect of uPA seems to be independent of its fibrynolytic activity.     

On the reversible interaction of plasminogen activator inhibitor-1 with tissue-type plasminogen activator and with urokinase-type plasminogen activator

The reaction between plasminogen activators and plasminogen activator inhibitor-1 is characterized by an initial rapid formation of an inactive reversible complex. The second-order association rate constant (k1) of complex formation of recombinant two-chain tissue-type plasminogen activator (rt-PA) or recombinant two-chain urokinase-type plasminogen activator (rtcu-PA) by recombinant plasminogen activator inhibitor-1 (rPAI-1) is 2.9 +/- 0.4 x 10(7) M-1 s-1 (mean +/- S.D., n = 30) and 2.0 +/- 0.6 x 10(7) M-1 s-1 (n = 12), respectively. Different molecular forms of tissue- or urokinase-type plasminogen activator which do not form covalent complexes with rPAI-1, including rt-PA-Ala478 (rt-PA with the active-site Ser478 mutagenized to Ala) and anhydro-urokinase (rtcu-PA with the active-site Ser356 converted to dehydroalanine) reduced k1 in a concentration-dependent manner, compatible with 1:1 stoichiometric complex formation between rPAI-1 and these ligands. The apparent dissociation constant (KD) of the complex between rPAI-1 and rt-PA-Ala478, determined as the concentration of rt-PA-Ala478 which reduced k1 to 50% of its control value, was 3-5 nM. Corresponding concentrations of active-site-blocked two-chain rt-PA were 150-250-fold higher. The concentration of anhydro-urokinase which reduced k1 to 50% was 4-6 nM, whereas that of active-site-blocked rtcu-PA was 100-250-fold higher. Recombinant single-chain urokinase-type plasminogen activator had an apparent KD of about 2 microM. These results suggest that inhibition of rt-PA or rtcu-PA by rPAI-1 proceeds via a reversible high affinity interaction which does not require a functional active site but which is markedly reduced following inactivation of the enzymes with active-site titrants.     

Target-selective activation of a TNF prodrug by urokinase-type plasminogen activator (uPA) mediated proteolytic processing at the cell surface

We have previously developed TNF prodrugs comprised of a N-terminal scFv targeting, a TNF effector and a C-terminal TNFR1-derived inhibitor module linked to TNF via a MMP-2 motif containing peptide, allowing activation by MMP-2-expressing tumor cells. To overcome the known heterogeneity of matrix metalloprotease expression, we developed TNF prodrugs that become processed by other tumor and/or stroma-associated proteases. These TNF prodrugs comprise either an uPA-selective or a dual uPA-MMP-2-specific linker which displayed efficient, target-dependent and cleavage sequence-specific activation by the corresponding tumor cell-expressed proteases. Selective pharmacologic inhibition of endogenous uPA and MMP-2 confirm independent prodrug processing by these two model proteases and indicate the functional superiority of a prodrug containing a multi-specific protease linker. Processing optimised TNF prodrugs should increase the proportion of active therapeutic within the targeted tissue and thus potentially enhance tumor response rate.Authors Jeannette Gerspach and Julia Németh have contributed equally to this work     

Bovine embryos produce a urokinase-type plasminogen activator.

The type of plasminogen activator (PA) secreted by bovine embryos was identified. Day 12-14 embryos were collected from estrus-synchronized, superovulated, and naturally mated crossbred beef cows. Embryos were left intact (E) or microdissected into component embryonic discs (ED) and trophoblastic vesicles (TV). Intact embryos, ED, and TV were pre-cultured for 2 days in Minimum Essential Medium Alpha (MEM alpha) with 10% heat-inactivated fetal calf serum, washed in serum-free MEM alpha, and cultured individually for 5 days in 50 microliters microdrops of MEM alpha with 15 mg/ml bovine serum albumin. At 24 hr intervals, E, ED, and TV were observed for tissue morphology and transferred to fresh microdrops, and medium was recovered and frozen at -20 degrees C. At the end of culture, blastocoelic fluid (BF) and embryonic tissues were recovered and frozen at -20 degrees C. Plasminogen activator concentrations in medium, tissues, and BF were determined by using a caseinolytic assay. Antibodies to urokinase-type PA (anti-uPA) and tissue-type PA (anti-tPA), and the urokinase inhibitor, amiloride (AMR), were used to identify the type of PA produced by bovine embryonic tissues. Intact embryos and TV released more PA (P less than 0.05) than ED, and tissues exhibiting expanded blastocoels released less PA (P less than 0.05) than tissues with collapsed blastocoels. Blastocoelic fluid from TV exhibited more PA (P less than 0.05) activity than from ED. Treatment with anti-uPA decreased PA activity (P less than 0.05) in pooled medium and tissues from E compared to treatment with nonspecific immunoglobulins and anti-tPA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Concentrations of plasminogen activator inhibitor-1 (PAI-1) and urokinase plasminogen activator (uPA) in induced sputum of asthma patients after allergen challenge

Urokinase plasminogen activator (uPA) and its inhibitor (PAI-1) are involved in tiisue remodeling and repair processes associated with acute and chronic inflammation. The aim of the study was to evaluate the effect of allergen challenge on concentration of uPA and PAI-1 in induced sputum of house dust mite allergic asthmatics (HDM-AAs). Thirty HDM-AAs and ten healthy persons (HCs)were recruited for the study. In 24 HDM-AAs bronchial challenge with Dermatophagoides pteronyssinus (Dp) and in 6 HDM-AAs sham challenege with saline were performed. In HDM-AAs sputum was induced 24 hours before (T0) and 24 hours (T24) after the challenge. Concentration of uPA and PAI-1 in induced sputum were determined using immunoenzymatic assays. At T0 in HDM-AAs mean sputum uPA (151 ± 96 pg/ml) and PAI-1 (4341 ± 1262 pg/ml) concentrations were higher than in HC (18.8 ± 6.7 pg/ml; p=0.0002 and 596 ± 180 pg/ml; p<0.0001; for uPA and PAI-1 respectively). After allergen challenge further increase in sputum uPA (187 ± 144 pg/ml; p=0.03) and PAI-1 (6252 ± 2323 pg/ml; p<0.0001) concentrations were observed. Moreover, in Dp challenged, but not in saline challenged HDM-AAs the mean uPA/PAI-1 ratio decreased significantly at T24. No significant increase in the studied parameters were found in sham challenged patients. In HDM-AAs allergen exposure leads to activation of the plasmin system in the airways. Greater increase of the PAI-1 concentration than uPA concentration after allergen challenge may promote airway remodeling and play an important role in the development of bronchial hyperreactivity.     

Urinary-type plasminogen activator (uPA) expression and uPA receptor localization are regulated by alpha 3beta 1 integrin in oral keratinocytes

Expression of urinary-type plasminogen activator (uPA) and its receptor (uPAR) is correlated with matrix proteolysis, cell adhesion, motility, and invasion. To evaluate the functional link between adhesion and proteolysis in gingival keratinocytes (pp126), cells were treated with immobilized integrin antibodies to induce integrin clustering. Clustering of alpha(3) and beta(1) integrin subunits, but not alpha(2), alpha(5), alpha(6), or beta(4), enhanced uPA secretion. Bead-immobilized laminin-5 and collagen I, two major alpha(3)beta(1) ligands, also induced uPA expression. Coordinate regulation of the serpin plasminogen activator inhibitor 1 was also apparent; however, a net increase in uPA activity was predominant. alpha(3)beta(1) integrin clustering induced extracellular signal-regulated kinase 1/2 phosphorylation, and both uPA induction and extracellular signal-regulated kinase activation were blocked by the mitogen-activated protein kinase/extracellular signal-regulated kinase kinase inhibitor PD98059. Integrin aggregation also promoted a dramatic redistribution of uPAR on the cell surface to sites of clustered alpha(3)beta(1) integrins. Co-immunoprecipitation of beta(1) integrin with uPAR provided further evidence that protein-protein interactions between uPAR and beta(1) integrin control uPAR distribution. As a functional consequence of uPA up-regulation and uPA-mediated plasminogen activation, the globular domain of the laminin-5 alpha(3) subunit, a major pp126 matrix protein, was proteolytically processed from a 190-kDa form to a 160-kDa species. Laminin-5 containing the 160-kDa alpha(3) subunit efficiently nucleates hemidesmosome formation and reduces cell motility. Together, these data suggest that multivalent aggregation of the alpha(3)beta(1) integrin regulates proteinase expression, matrix proteolysis, and subsequent cellular behavior.     

3D-QSAR CoMFA/CoMSIA studies on Urokinase plasminogen activator (uPA) inhibitors: a strategic design in novel anticancer agents

Comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) was performed on a series of indole/benzoimidazole-5-carboxamidines as urokinase plasminogen activator (uPA) inhibitors. The ligand molecular superimposition on template structure was performed by atom/shape-based RMS fit, multifit, and RMSD fit methods. The removal of two outliers from the initial training set of 30 molecules improved the predictivity of the models. The statistically significant model was established from 28 molecules, which were validated by evaluation of test set of nine compounds. The atom-based RMS alignment yielded best predictive CoMFA model (r2(cv) = 0.611, r2(cnv) = 0.778, F value = 43.825, r2(bs) = 0.842, r2(pred) = 0.616 with two components) while the CoMSIA model yielded (r2(cv) = 0.499, r2(cnv) = 0.976, F value=96.36, r2(bs) = 0.993, r2(pred) = 0.694 with eight components). The contour maps obtained from 3D-QSAR studies were appraised for the activity trends of the molecules analyzed. The results indicate that the steric, electrostatic, and hydrogen bond donor/acceptor substituents play significant role in uPA activity and selectivity of these compounds. The data generated from the present study can be used as putative pharmacophore in the design of novel, potent, and selective urokinase plasminogen activator inhibitors as cancer therapeutics.     

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.