[Frontiers in Bioscience 14, 3782-3794, January 1, 2009]

Structure, function and antagonists of urokinase-type plasminogen activator

Maria Vincenza Carriero1, Paola Franco2, Imma Vocca2, Daniela Alfano2, Giuseppina Votta2, Immacolata Longanesi-
Cattani1, Katia Bifulco1, Alessandro Mancini3, Mario Caputi3, Maria Patrizia Stoppelli2
1
National Cancer Institute, Via Semmola, Naples, 2 Institute of Genetics and Biophysics “Adriano Buzzati-Traverso”, National
Research Council, Naples, 3 Department of Cardio-Thoracic Sciences, Second University of Naples, Naples, Italy

TABLES OF CONTENTS

1. Abstract
2. Introduction
3. The multidomain protease urokinase
3.1. The growth factor-like domain
3.2. The kringle domain
3.3. The connecting peptide region
3.4. The catalytic domain
4. Functional properties of urokinase
4.1. Regulatory mechanisms of urokinase synthesis, localisation and activity
4.2. Urokinase and human pathology
5. Antagonists to urokinase
5.1. Antagonists of growth factor-like domain
5.2. Antagonists kringle domain and connecting peptide region
5.3. Inhibitors of urokinase enzymatic activity
5.4. Inhibitors of urokinase expression
6. Perspectives
7. References

1. ABSTRACT 2. INTRODUCTION

Urokinase (uPA) is a serine protease which The serine protease urokinase (uPA) is secreted
converts plasminogen to plasmin, a broad-spectrum as a 411 aminoacids single-chain zymogen form (pro-uPA)
protease active on extracellular matrix (ECM) components. which becomes activated by plasmin cleavage in the
Like many components of the blood coagulation, extracellular milieu (1). In turn, active uPA converts
fibrinolytic and complement cascades, uPA has a modular inactive plasminogen into the active serine protease
structure, including three conserved domains: a growth plasmin, able to degrade most of the ECM components and
factor-like domain (GFD, residues 1 – 49), a kringle activate latent collagenases and growth factors. Pro-uPA
domain (residues 50 – 131), linked by an interdomain consists of a growth factor-like domain (GFD, residues 1 –
linker or “connecting peptide” (CP, residues 132 – 158) to 49), a kringle domain (residues 50 – 131), an interdomain
the serine protease domain (residues 159 – 411). Although linker or “connecting peptide” (CP, residues 132 – 158),
direct molecular interactions with urokinase receptor and and a serine protease domain (residues 159 – 411) (Figures
integrins have been extensively described, the function of 1 and 2). Activation of the pro-enzyme occurs by
single uPA domains is not completely understood. Because proteolytic cleavage at Lys 158-Ile159, thus generating a two-
of the causal involvment of uPA in cancer invasion and chain molecule linked by a disulfide bridge between Cys148
metastasis, the blockade of uPA interactions and activity and Cys279 (HMW uPA). A further proteolytic cleavage
with specific inhibitors is of interest for novel strategies in releases an amino-terminal fragment (ATF, residues 1 –
cancer therapy. New inhibitors derived from the 135) and a small region linked to a large C-terminal
interdomain linker or “connecting peptide” are coming into proteolytic domain (LMW uPA) (2). In vivo, pro-uPA may
focus. This review summarizes the recent findings on the be activated by plasmin and also by other proteases,
uPA structure-function relationship and provides further including glandular kallikrein mGK-6, as shown by the
information on existing inhibitors of uPA multiple analysis of urine from plasminogen-deficient mice (3).
functions. Hepsin is an example of type II transmembrane protease

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exons encoding single functional domains were exchanged

between different genes by intronic recombination events
(8, 14). This observation well agrees with the finding that
single uPA domains are structurally and functionally
autonomous (15).

3. THE MULTI-DOMAIN PROTEASE UROKINASE

3.1. The growth factor-like domain

Like bood coagulation factors VII, IX and X and
the anticoagulant protein C, uPA features a conserved
growth factor-like module which is homologous to the
EGF-like protein family (16). The EGF-like domain
includes six cysteine residues which are engaged in
disulfide bonds. The structure of several EGF-like
domains has been solved. In all cases, a two-stranded
beta-sheet is followed by a loop and a C-terminal short
two-stranded sheet (17). Consolidated evidence shows that
GFD mediates the binding of uPA to a
glycosylphosphatidylinositol (GPI)-anchored cell surface
molecule, also known as uPAR, which was discovered over
twenty years ago (18-19). The receptor binding ability of
uPA is retained by a peptide corresponding to residues 12-
32 of the human sequence (16). A mutagenic analysis of
the residues involved in the full molecule shows that
Lys23, Tyr24, Phe25, IIe28, and Trp30 are essential for
uPAR binding (20). Recent evidence reports the occurrence
of intra-molecular interactions between kringle and GFD
which stabilize uPA/uPAR complexes. This is supported by
Figure 1. The 3-dimensional structure of human uPA. The the fact that a pro-uPA variant lacking the kringle domain
figure shows the catalytic triad Ser195, His57, and Asp102, binds to soluble uPAR with a similar "on-rate" but with a
and Asp189 in the S1 pocket (pink); the main chain binding faster "off-rate" than wild-type pro-uPA (21). Pro-uPA,
area around residues 214-217 and 41 (green); and uPA's uPA and ATF exhibit a similar KD for binding to uPAR, in
specific surface loops (red) (Reproduced with permission the range of 100 - 500 pM (22). Receptor-bound uPA is
of P.A. Andreasen). proteolytically active and can be inhibited by plasminogen
activator inhibitor type-1 (PAI-1). As shown by kinetic
which is able to convert pro-uPA into an active studies, cell surface plasminogen activation exhibits an
enzyme (4). Pro-uPA may undergo several post- overall increase of kcat/Km of 5.7-fold with respect to the
translational modifications, such as glycosylation of Asn302, same reaction with soluble components. Therefore,
phosphorylation on Ser138/303 and fucosylation of Thr18 (5- receptor-bound uPA endows cells with an efficient matrix-
6). In particular, the latter two modifications modulate the degrading ability (23). Although it is not the subject of this
catalytically-independent chemotactic and mitogenic review, most of the described biological outcomes
activities of uPA, respectively (5, 7). Pro-uPA is encoded following uPA/uPAR association are proteolytically
by a 6.4 kb gene which is located on the long arm of independent and relate to cell migration, adhesion and
chromosome 10 (8-9). The TATA-box containing minimal proliferation (24). Remarkably, the mitogenic activity of a
promoter ensures a low level of uPA basal expression and peptide corresponding to residues 4 – 43 and full-length
includes a GC-rich region and a CAAT sequence binding to uPA on human SaOS-2 osteosarcoma cells is dependent on
the ubiquitous factors Sp1 and CTF, respectively. An fucosylation of Thr18. On the contrary, motogen and anti-
upstream enhancer is located at –2 kb and requires the apoptotic activities of uPA are retained by GFDp, a peptide
cooperation between an Ets(PEAIII)/AP1 and an AP1 site corresponding to the amino-acids 12-32, showing that post-
to up-regulate uPA mRNA transcription (10). This translational modifications in this region are not relevant to
cooperation is mediated by a 74-bp protein-binding domain, these functions (25-26).
the cooperation mediator element (11). It is noteworthy that
transcription factors binding to the Ets/AP-1 sites are 3.2. The kringle domain
activated by the Ras/MAPK-dependent pathway, which is Kringles are autonomously folding domains
triggered by a variety of stimuli like growth factors, retaining similar architecture during evolution. A likely
cytokines, uv light and is hyper-activated in most tumors possibility is that kringles of prothrombin, plasminogen,
(12). Transcriptional regulation may be mediated also by tissue plasminogen activator, coagulation factor XIIa and
the NF-kB complex acting through binding of Rel/p65 and uPA may have evolved from a common ancestral module
p65/p50 to a specific sequence located at –1583 bp (13). (27). The uPA kringle and kringle 2 of tissue-type
Interestingly, the overall analysis of the exon-intron plasminogen activator (tPA) domains exhibit similar global
organization of the pro-uPA gene strongly suggests that folds (28). Unlike the tPA kringle 2 which exhibits a lysine

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Figure 2. Interactions and functional effects of the urokinase single domains. Urokinase may affect cell physiology by a variety
of molecular mechanisms: first of all through its non-catalytic amino-terminal region (consisting of a growth factor-like domain
or GFD, a kringle domain and a “connecting peptide” or CP) which interacts with uPAR and integrins. Second, through a
proteolytically-dependent mechanism by cleaving, at least, three substrates like plasminogen, pro-MSP (precursor to macrophage
stimulating protein) and pro-HGF (pro-hepatocyte growth factor). The catalytic activity of uPA results in fibrin and matrix
degradation as well as in the direct, or plasmin-mediated, activation of several growth factors, thus affecting cell migration,
adhesion, scattering, survival/apoptosis and proliferation.

binding site, isolated kringle fragments of uPA bind 144 have been observed in the lungs of mice exposed to
polyanions like heparin trough a basic cluster of Arg108, LPS which appear to be mediated by Akt and NF-kB
Arg109, and Arg110 (29). By phage display technology, a activity (34). The search for cryptic activities of uPA which
putative uPA kringle-binding consensus sequence could be applied to anti-cancer therapy shows that FGF-
BXXSSXXB (where B represents a basic amino-acid and X stimulated proliferation and VEGF-induced migration of
represents any amino-acid), was identified and endothelial cells may be prevented by human recombinant
subsequently found in the gp130 signal transducer protein kringle domain (UK1, residues 45 – 135), possibly via an
(30). Interestingly, the uPA kringle binds to (alpha)v(beta)3 unknown uPAR-independent mechanism (35).
integrin and promote migration, which can be blocked by
plasminogen kringles 1-3 or 1-4 (angiostatin), a known 3.3. The connecting peptide region
integrin antagonist (31). Pluskota et al. report that pro-uPA Evidence for a role of the connecting peptide was
binding to (alpha)M(beta)2 integrin is critical for the originally suggested by the finding that uPA may be
(alpha)M(beta)2-mediated enhancement of plasmin phosphorylated on Ser138 and/or Ser303 with clearcut
generation. Interestingly, this binding was lost by a kringle- functional consequences (5). This post-translational
less pro-uPA mutant (lacking amino-acids 47 – 135), modification occurs in A431 human carcinoma cells prior
showing that kringle is required for this interaction (32). to secretion and is inhibited by protein kinase C inhibitors
Another study reports that an isolated uPA fragment (36). Phosphorylated uPA (P-uPA) retains full catalytic
corresponding to residues 43 – 156 promotes smooth ability and uPAR binding whereas it lacks the ability to
muscle cell migration at nanomolar concentrations in a stimulate cell adhesion and migration. Substitution of the
uPAR-independent manner (33). Acute pro-inflammatory critical Ser with Glu residues results in uPA variants with
effects of the mouse uPA region spanning residues 48 – weak or no chemotactic activity (5). Interestingly, Ser138 is

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conserved among different mammalian species, like mouse, removing D1 domain and exposing uPAR SRSRY
rat, yellow baboon, bovine and orangutan, suggesting a chemotactic epitope, which interacts with fMLP receptors
relevant functional role (Votta and Stoppelli, unpublished). (43). Most of the proteolytically driven effects of uPA are
Further evidence indicating the relevance of the connecting mediated by plasmin which may down-regulate the local
peptide region is based on the effects of Å6, a peptide concentration of fibrin, fibronectin, laminin, vitronectin and
corresponding to uPA residues 135-143 which inhibits proteoglycans. In addition, plasmin activation of latent
tumor progression and angiogenesis (37). Recently, an metalloproteases leads to collagen degradation (38). In
isolated peptide corresponding to uPA residues 135-158 vascular pathologies, such as the rupture of aortic
was reported to bind to (alpha)v(beta)5 integrin with high aneurysms, plasmin is an important activator of pro-
affinity, to induce chemotaxis at picomolar concentrations, metalloproteases (44). A previously unrecognized cellular
and to stimulate the association of uPAR and effect of the degradation of ECM molecules is cell
(alpha)v(beta)5 integrin (26). Interestingly, this binding was detachment, which may be followed by anoikis
not dependent on the GFD-uPAR interaction, but (corresponding to detachment-induced apoptosis). In this
potentiates the functional effects of GFD on chemotaxis. respect, the generation of plasmin activity may be pro-
The most ready interpretation of these observations is that apoptotic, whereas the expression of inhibitors, such as
the linker of uPA induces a conformational change in protease nexin-1 may favour cell survival (45). A novel
(alpha)v(beta)5 integrin, which impacts on cell migration role for plasmin in the differentiation of pancreatic
and invasion. These findings suggest that uPA may affect precursor cells has been proposed, raising the possibility
cell function by bridging uPAR and integrins, thus fully that islet formation by pancreatic precursor cells is
stimulating migration. triggered by plasminogen activation and regulated by
endogenous PAI-1 (46). Plasmin activity may also subvert
3.4. The catalytic domain growth factor signalling. In fact, following ECM
The crystal structure of the uPA catalytic domain degradation there is an increased availability of active
has been determined and shows that the enzyme has the bFGF and VEGF as well as the direct activation of latent
expected topology of a trypsin-like serine protease. The TGF-beta (47). In vivo, the multiple effects of plasmin
enzyme has an S1 specificity pocket, an S2 hydrophobic activity emerge from the analysis of plasminogen-deficient
pocket and an S3 pocket; insertions of extra-residues in mice, which survive embryonic development but are
loop regions create unique surface areas. A specific triad, impaired in growth, fertility and survival. In the same mice,
His 204, Asp 255, and Ser 356 is responsible for the skin wound healing is severely impaired, suggesting that
catalytic mechanism (17). The protease-specific surface plasmin activity is also required for tissue regeneration
loops surrounding the active site are also involved in (48). In humans the absence of plasminogen is associated to
exosite interactions of uPA with its substrates. It has long a pathological condition, known as ligneous conjunctivitis.
been known that active uPA catalyzes the conversion of the In non-severe cases, the application of plasminogen-
inactive zymogen plasminogen to the broad-spectrum containing eye drops may reduce symptoms (49).
protease plasmin by cleaving an R560-V561 peptide bond
(38). However, uPA can also activate the precursor to the 4. FUNCTIONAL PROPERTIES OF UROKINASE
scatter factor/hepatocyte growth factor pro-HGF, which
exhibits extensive homology with plasminogen, and the 4.1. Regulatory mechanisms of urokinase synthesis,
macrophage stimulating protein, thereby indirectly localisation and activity
controlling cell proliferation, ECM invasion and prevention The expression level, activity and localisation of
from apoptosis (39). There are two uPA inhibitors, namely uPA is strictly controlled in a variety of physiological and
PAI-1 and PAI-2 (plasminogen activator inhibitor type-2), pathological conditions. Expression of uPA may be
belonging to the serpin (serine protease inhibitors) family. regulated at transcriptional level through the activity of its
PAI-1 reacts more quickly that PAI-2 with uPA and causes promoter region and at the level of mRNA stability through
a non-reversible inhibition of the enzyme (38). Covalent mRNA-protein interactions. As described in the
uPA-PAI-1 complexes are cleared by uPAR-dependent Introduction, the uPA promoter region is highly responsive
internalization, if the protease is receptor-bound (40-41). to phorbol esters, growth factors, steroid hormones,
cytokines and uv light through the Ras/MAPK-dependent
Receptor-mediated membrane localization of pathway impinging on the Ets/AP-1 binding sites (12). In
uPA is of relevance to the proteolytic efficiency of the breast carcinomas, the Ets-1 and Ets-2 transcription factors
whole system. In particular, kinetic studies have shown that bind to uPA and MMP-9 promoters, in response to
receptor-bound uPA exhibits a 40-fold lower Km than epidermal growth factor, thus enhancing invasion and
soluble uPA and a concurrent 6-fold reduction in kcat, thus metastasis (50). The important role of histone modifications
resulting in an overall increase of catalytic efficiency (23). in regulating uPA gene expression is established by recent
Since plasminogen can become membrane-bound, the findings highlighting the induction of uPA expression by
occurrence of receptors for plasminogen and uPA on the histone deacetylase inhibitors trichostatin A and sodium
same cell results in the enhanced formation of surface- butyrate in human cancer cells (51). uPA expression may
associated plasmin. This machinery generates broad- be controlled also at the level of mRNA stability by many
spectrum proteolytic activity, which is restricted to cell factors, such as protein synthesis inhibitors, PKC activity,
surface and protected by circulating inhibitors, such as glucocorticoids. This regulatory mechanism involves one
(alpha)2-antiplasmin (42). The proteolytic activity of (AU)-rich sequence within the 3’-UTR of uPA mRNA. The
receptor-bound uPA may contribute to signaling by (AU)-rich sequence-dependent degradation mechanism is

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impaired in breast carcinoma cell lines and the half-life of inflammation and tissue repair. For example, uPA-/- mice
uPA mRNA is markedly longer than in normal cells (52). had a severe regeneration defect, with decreased
Constitutive expression of uPA leading to a highly recruitment of blood-derived monocytes to the site of injury
metastatic phenotype is achieved by an increase in mRNA and with persistent myotube degeneration. In addition,
stability of MDA-MB-231 breast carcinoma cells (53). At uPA-deficient mice accumulated fibrin in the degenerating
molecular level, phosphorylation of mitogen-activated muscle fibers (61). Acute viral myocarditis is an important
protein kinase-activated protein kinase 2 (MK2) results in cause of cardiac failure in humans. In mouse models of
an enhanced binding of HuR proteins to the AU-rich acute viral myocarditis, it has been observed an increased
elements in the 3' untranslated regions of uPA and uPAR, expression of uPA and MMP-9. Interestingly, loss of uPA
thus leading to mRNA stabilization (54). Among the or MMP activity reduces the cardiac inflammatory
factors controlling uPA synthesis is LPA (lysophosphatidic response, thereby protecting against cardiac injury,
acid), which is an important intercellular signaling dilatation, and failure during virally-induced myocarditis
molecule participating to pathogenesis of many human (62).
diseases. LPA upregulates uPA secretion via p38(MAPK)
signaling pathway in ovarian cancer cells (55). uPA Available data support the hypothesis that
increased expression may be also a consequence of a plasminogen could play a role in certain skin diseases.
decreased cell-cell adhesion. Disruption of E-cadherin- Transgenic mice overexpressing uPA and uPAR in basal
dependent cell-cell adhesion by a soluble E-cadherin epidermis develop extensive alopecia due to involution of
fragment or by the function-blocking antibody Decma hair follicles, epidermal thickening and sub-epidermal
results in Src-dependent Erk activation and in uPA blisters, a phenotype due to receptor-bound uPA catalytic
induction (56). activity (63).
The regulation of uPA localization is achieved by The plasminogen activation system represents a
binding to uPAR which focalizes protease activity on cell potent mechanism of extracellular proteolysis and is an
surface and mediates uPA-dependent signaling (23). Early essential component of normal wound healing, as shown in
results showed that in human fibroblasts receptor-bound mice models. In particular, a functional cooperation
uPA is mainly found at focal contact sites, suggesting that between the plasminogen/plasmin and the metalloprotease
uPA may restrict uPAR lateral mobility (57). UPAR cascade is suggested by the finding that complete inhibition
clustering is induced in differentiating myelomonocytic U937 of the healing process requires both plasminogen
cells by pre-incubation with uPA or ATF (58). The enzymatic deficiency and metalloprotease inhibition (64). Urokinase
activity of soluble and receptor-bound uPA may be controlled is regarded as an important trigger of the fibrinolytic
by the specific soluble inhibitors PAI-1 (plasminogen activator process, via plasminogen activation. Although uPA-
inhibitor type 1) and PAI-2. (plasminogen activator inhibitor deficient mice do not have major thrombotic disorders,
type-2). Although both molecules are strong inhibitors of uPA double uPA–tPA-knockout mice show extensive
proteolytic activity, they seem to play additional roles in cell extracellular fibrin deposition very similar to that observed
biology. First of all, PAI-1 binds with high affinity to matrix in plasminogen-knockout mice (65). One evidence is that
vitronectin, thus affecting cell adhesion and is reported to be uPA expression in atherosclerotic arteries contributes to
anti-apoptotic. Remarkably, PAI-1 is one of the most intimal growth and constrictive remodeling leading to
informative biochemical markers of a poor prognosis in lumen loss, thus accelerating atherogenesis. Antagonists of
several human cancer types. Unexpectedly, recent studies on uPA activity might, therefore, be useful in limiting intimal
cells expressing PAI-1 in human tumors and data generated in growth and preventing constrictive remodeling (66).
PAI-1 deficient mice support the possibility that PAI-1 Interestingly, in a mouse model of glomerulonephritis, a
promotes tumor growth and spread (59). Regarding PAI-2, the reduced uPA-mediated proteolysis correlates with
fact that only a small percentage of PAI-2 is secreted argues excessive fibrin deposition (67). In the case of infection,
for alternative roles of this serpin. Consistently with this uPA might first favor the recruitment and activation of
observation, PAI-2 alters gene expression, influences the rate immune cells, activate latent pro-inflammatory cytokines,
of cell proliferation and differentiation, and inhibits apoptosis and modulate T-lymphocyte responses (68). Mice deficient
in a uPA-independent manner (60). in uPA fail to generate type 1 immune responses during
infection with Cryptococcus neoformans (69). Data show
4.2. Urokinase and human pathology that the uPA-deficient mice are more susceptible than
Consolidated evidence shows that uPA has a controls to botryomycosis, a staphylococcal infection,
relevant role in many physiological conditions such as suggesting that uPA contributes significantly to the
intravascular fibrinolysis, angiogenesis, tissue regeneration immune response (70).
and immune response. Different pathological conditions
turned out to causally involve a dysregulated production of Urokinase has been shown to play crucial roles in
uPA, such as cancer growth and metastasis. The deep tumor progression as a soluble or membrane-associate
molecular knowledge of the uPA system is helping to protease. Experiments with cell cultures and animal models
define the molecular players acting under these have strongly indicated a pivotal biological role of uPA-
pathological conditions. catalysed plasminogen activation in tumor progression (1,
2, 38). Most recently, the results from studies involving
The activity of uPA may be required for an uPA-deficient mice (uPA-/- mice) were all in agreement
efficient tissue regeneration in diseases involving with the idea that uPA-catalysed plasmin generation is rate-

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limiting for tumor growth, local invasion and metastasis Given the crucial role of the uPA/uPAR
(71-72). In principle, a high level of uPA in extracts of interaction in tumor invasion and metastasis, specific
primary breast carcinomas was reported to predict an early antagonists can be regarded as cancer therapeutics. Early
relapse (73-74). Consistent with this possibility, the evidence showed that a peptide corresponding to residues
expression level of uPA and uPAR in breast carcinomas are 18 – 32 of the human uPA is the minimal sequence
higher than in the normal counterparts (75-76). This allowing binding to uPAR (16). More recently, Magdolen
relationship was later confirmed with other cancer types et al. have identified a synthetic cyclic peptide covering the
(77) The concept of uPAR-dependent pericellular residues 19–31 (cyclo19,31[D-Cys19] peptide) as a potent
proteolysis in breast cancer is supported by the finding that inhibitor of uPA/uPAR association (82). However, it has to
fibroblast-derived stromal uPA enhances epithelial tumor be taken into consideration that peptides spanning the
cell invasion by up-regulating uPA and (alpha)v(beta)5 uPAR binding domain, like GFD, may stimulate cell
vitronectin receptors (78). The role of uPA should be migration and signalling at nanomolar concentrations (26).
considered in view of the cross-talk of primary tumors with An alternative approach is directed to the inhibition of the
the surrounding tumor stroma, which in different tumors uPA-uPAR interaction using small synthetic peptide
has been shown to secrete uPA and MMPs (79). The antagonists isolated by combinatorial chemistry. Å 9-mer
hypothesis of the importance of stromal uPA is consistent peptide was a potent inhibitor of uPAR binding and
with the findings that a genetically mammary gland tumor, reduced the intra-vasation of HEp-3 cancer cells by approx.
in which uPA is expressed mainly by stromal cells, 60% in a chicken chorioallantoic membrane assay (83).
disseminated slower in uPA-/- mice than in wild type mice
(72). In vivo, the proteolytic activity of plasmin seems to be 5.2. Antagonists of kringle domain and connecting
required for Polyoma T-induced vascular tumor formation, peptide region
as the combined loss of uPA and tPA leads to a One of the first monoclonal antibodies directed to
significantly reduced tumor growth rate (80). Indirect uPA recognizes an epitope located between GFD and
evidence that uPA proteolytic activity is relevant to tumor kringle domains and has been widely employed for affinity
angiogenesis is provided by studies in mice deficient in the purification of uPA, ATF and pro-uPA (84). Although 5B4
PAI-1 showing that PAI-1 is pro-angiogenic at monoclonal antibody reduces uPAR binding, it is unknown
physiological concentrations through its anti-proteolytic whether it affects uPA-integrin interaction. Anti-kringle
activity (81). In the emerging picture, the uPA has the monoclonal antibodies which block the binding of uPA to
ability to support the malignant phenotype through several polyanions have been subsequently developed (29). Given
mechanisms. First of all, by virtue of its matrix degrading the recent reports showing the association of the kringle
ability, which favors tumor dissemination; second, by and connecting peptide regions with several integrins, it
stimulating cell motility through the association with uPAR would be interesting to assess whether any of these
and integrins; third, by stimulating cell proliferation and by monoclonals may block these interactions and the relative
protecting cells from apoptosis, thus enhancing tumor cell functional effects. The chemotactic and integrin-binding
survival in a uPAR-dependent manner. The uPAR itself, abilities of the uPA interdomain linker region emerged
other than concentrating uPA proteolytic activity on cell quite recently, both in the full length molecule and in the
surface and being a mediator of most ligand-dependent isolated form, as CPp (residues 135-158) (26). Previous
effects on growth, motility and apoptosis, is itself an anti- evidence was based on the analysis of the uPA serine
apoptotic factor (25). This information support the phosphorylation occurring on Ser138, located in the CP
possibility that dysregulation of uPA level and/or region. Interestingly, Glu-substituted variants are
localisation might cause or favor different diseases, and chemotactically inactive and may be considered antagonists
suggest that interference with uPA-activity could block of the linker region (5). More recent evidence shows that
disease. More remains to be elucidated to understand how uPAS138E and uPAS138E/303E prevent migration induced by
to manipulate the uPA–uPAR system in pathology, without wild type uPA and by other chemoattractants (Franco,
hampering their physiological function in fibrinolysis and Vocca and Stoppelli, unpublished).
tissue remodeling.
The activity of peptides corresponding to
5. ANTAGONISTS TO UROKINASE sequences of the connecting peptide region is exemplified
by Å6, a 8-mer capped peptide (residues 135 – 143). This
In principle, there are several ways to interfere peptide inhibits tumor cell invasion as well as angiogenesis
with uPA function: one approach is the identification of in vitro and in vivo (37). Å6 has been reported to inhibit
inhibitors blocking the protease catalytic activity with high dissemination of Mat B-III rat breast cancer cells and
affinity and selectivity. Another possibility is to antagonise formation of new blood vessels, the effects being greater in
the interaction of GFD with the uPAR. A third possibility is the presence of Å6 and tamoxifen (85). Similarly, Å6 has
to interfere with uPA-integrin interaction, which has been been shown to inhibit growth and neo-vascularisation of
recently described as a property of the kringle and human U87MG glioblastoma cells growing on nude mice,
connecting peptide regions. Finally, uPA mRNA particularly when combined with cisplatin (86).
expression could be targeted by the use of RNA Furthermore, Å6 has been reported to suppress retinal neo-
interference technology. vascularisation in an animal model (87). The mechanism of
action of Å6 has not been fully elucidated. Surface plasmon
5.1. Antagonists of growth factor-like domain resonance analyses showed that Å6 acts at 1x10-6M
concentrations and may interfere with the uPA-uPAR

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interaction (37). However, recent data show that that CPp Taken together, these findings suggest that uPA
(corresponding to residues 135-158 of human uPA) retains enzymatic activity may be controlled in vivo by many
the uPA ability to associate with (alpha)v(beta)5 integrin at potent and selective inhibitors which may be employed in
nanomolar concentrations, suggesting that Å6 may act the control of tumor dissemination, wound healing and
through integrins (26). However, further experiments are other pathologies caused by excess uPA activity. Finally,
needed to clarify this issue. Recently, a phase I clinical trial an interesting possibility is to take advantage of uPA
of Å6 in gynaecologic malignancies has been completed activity to target cancer cells. Recently, an engineered
and indicated neither toxicity nor any immunogenic anthrax toxin that is selectively activated by cell-surface
response (88). uPA was generated. The novel recombinant anthrax toxin,
PrAgU2/FP59, composed of the uPA-activated protective
5.3. Inhibitors of enzymatic activity antigen and a fusion protein of Pseudomonas exotoxin and
Proteolytic activity generated by uPA is relevant lethal factor showed anti-lung cancer efficacy in an in vivo
to metastasis and angiogenesis. Thus, the inhibition of uPA human tumor model (105).
is a promising strategy in cancer. In the ‘80s, polyclonal
anti-uPA antibodies were obtained to prevent uPA activity 5.4. Inhibitors of urokinase expression
in vitro and in a chicken tumor model (89-90). High level expression of uPA has been well
Subsequently, several monoclonal antibodies recognizing documented in a variety of tumors and correlated to a poor
different epitopes in the uPA molecule were generated (91- prognosis. Therefore, inhibition of uPA expression may be
92). In the last ten years, many potent and selective regarded as an anti-tumor strategy. In recent years, RNA
molecules inhibiting the catalytic activity of uPA have been interference (RNAi) technology has been recognised as a
obtained. Available structural information of enzyme- post-transcriptional effective methodology to silence
inhibitor complexes has greatly accelerated the specific genes (106). In principle, the inhibition of uPA
optimisation process of in silico designed inhibitors (93). synthesis can be achieved by a stable transfection with an
These data were used to identify a class of uPA inhibitors antisense uPA vector or by transfection with small
based on the 2-naphthamidine template that exhibit a interfering RNA molecules (siRNA). According to Arens
remarkable selectivity for uPA over trypsin-like enzymes N. et al, the use of specific siRNAs provides a more
(94). Based on the peptide sequence of an optimal uPA efficient down-regulation of uPA mRNA than the
substrate, phenethylsulfonyl-D-Ser-Ala-D-aminomethyl introduction of an antisense uPA vector (107). Several
benzamidine was characterised as a selective uPA inhibitor groups have explored the use of siRNAs to target uPA
with a Ki value of 3.1 nM (95-96). A similar inhibitor and/or uPAR for therapeutic applications in cancer
prevented lung metastases and prolonged survival of nude treatment. An early report shows that SKHep1C3
mice injected with HT1080 fibrosarcoma cells (97). WX- epatocarcinoma cells transfected with a plasmid coding for
UK1, a novel inhibitor of uPA derived from 3- an RNA composed of two identical 19-nucleotide sequence
amidinophenylalanine, inhibits matrigel invasion of breast motifs forming an hairpin dsRNA showed a consistently
and cervical carcinoma cells as well as spreading of decreased level of uPA protein as well as a reduction of
orthotopically transplanted BN472 breast tumors in rats, migration, invasion, and proliferation (108). The
thus suggesting a novel adjuvant antimetastatic therapy simultaneous targeting of uPA and uPAR is a powerful
(98-99). Among the 1-isoquinolinylguanidines which are approach, as it results in a decrease in the phosphorylation
potent and selective inhibitors of uPA (Ki 10 nM), UK- of the Ras-activated pathway molecules such as FAK,
371,804 is the most characterised. Following local p38MAPK, JNK and ERK1/2, as well as the MEK-
administration, this compound effectively inhibited uPA in activated phosphatidylinositol 3-kinase (PI3k) pathway in
a porcine acute excisional wound model. In particular, UK- glioma cells (109). SKHep1C3 cells stably transfected with
371,804 is recommended in the treatment of chronic dermal a plasmid coding for an siRNA directed to human uPA
ulcers, which are characterised by excessive uncontrolled exhibited a consistent reduction of migration, invasion, and
proteolytic degradation resulting in ulcer extension, loss of proliferation (108). Another approach to silence uPA
functional matrix molecules (e.g., fibronectin), and expression is based on the fact that DNA demethylation is a
retardation of epithelialization (100). The uPA inhibitor common mechanism underlying the abnormal expression of
WXC-340 prevents metastatic growth following surgery in uPA. Histone deacetylase (HDAC) inhibitor trichostatin A
a murine colorectal carcinoma model, suggesting that stimulates uPA expression and cancer cell invasion, raising
perioperative uPA inhibition with WXC-340 may represent the possibility that HDAC inhibitors employed as cancer
a novel anti-tumor strategy (101). A 4-oxazolidinone therapeutics may favour metastasis by up-regulating uPA
analogue (UK122) which inhibits uPA activity with a Ki of (51). Inhibition of demethylation of uPA promoter with S-
0.2x10-6M was developed. UK122 significantly inhibits Adenosyl-l-methionine results in uPA silencing and
pancreatic cancer cell migration and invasion capability inhibition of MDA-231 cells tumor cell invasion in vitro
(102). By screening phage-displayed random peptide and tumor growth and metastasis in vivo (110-111).
libraries with uPA as the target, the disulfide bridged
sequence CSWRGLENHRMC (upain-1) was isolated Furthermore, nucleic acid molecules with high
(103). A similar strategy yielded a specific inhibitor for affinity for a target transcription factor can be introduced
mouse uPA (104). Although both molecules specifically into cells as decoy cis-elements to bind these factors and
inhibit plasminogen activation, their effect in mouse cancer alter gene expression. For instance, a peptide nucleic acid
models remains to be investigated. chimera containing an Sp1 binding sequence, has been
investigated to down modulating the expression of Sp1

3788
Multidomain protease urokinase and its inhibitors

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Abbreviations: uPA, Urokinase Plasminogen Activator;

ECM, Extra Cellular Matrix; pro-uPA, single-chain zymogen
form of uPA; uPAR, Urokinase Receptor; CP, uPA connecting
peptide region (residues 132-158); GFD, uPA growth factor-
like domain (residues 1-49); ATF, Amino-Terminal Fragment;
GPI, glycosylphosphatidylinositol; P-uPA Phosphorylated

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