Tripeptides with C-terminal arginine as potential inhibitors of urokinase

Article abstract of DOI:10.1007/s10989-011-9239-y

We synthesized and tested ten peptides with the molecular structure being H-d-Ser-AA-Arg-OH for their effect on the amidolytic activities against urokinase, thrombin, trypsin, plasmin, tissue plasminogen activator and kallikrein. The inserted amino acid in each peptide was either leucine, norleucine, izoleucine, valine, norvaline, α-metyloalanine, α-aminobutanoic acid, homoleucine, tert-leucine or neoglycine. H-d-Ser-NVal-Arg-OH (compound 4) was the most active inhibitor of urokinase plasminogen activator with a K_i value of 0.85 μM. Compound 4 showed cytotoxic effect against MDA-MB-231 and DLD cell lines, respectively, with IC₅₀ values of 25 and 19 μM. Synthesised compounds did not have activity against MCF-7 cancer cells. These peptides were nontoxic against pig's erythrocytes in vitro.

Full text of DOI:10.1007/s10989-011-9239-y

Int J Pept Res Ther (2011) 17:47–52
DOI 10.1007/s10989-011-9239-y
SHORT COMMUNICATION
Tripeptides with C-Terminal Arginine as Potential Inhibitors
of Urokinase
•
Agnieszka Markowska Irena Bruzgo
•
•
Wojciech Miltyk Krystyna Midura-Nowaczek
Accepted: 5 January 2011 / Published online: 15 January 2011
Ó Springer Science+Business Media, LLC 2011
Abstract We synthesized and tested ten peptides with the
molecular structure being H–D-Ser–AA–Arg–OH for their
effect on the amidolytic activities against urokinase,
thrombin, trypsin, plasmin, tissue plasminogen activator
and kallikrein. The inserted amino acid in each peptide was
either leucine, norleucine, izoleucine, valine, norvaline,
a-metyloalanine, a-aminobutanoic acid, homoleucine,
tert-leucine or neoglycine. H–D-Ser–NVal–Arg–OH (com-
pound 4) was the most active inhibitor of urokinase plas-
minogen activator with a Ki value of 0.85 lM. Compound
4 showed cytotoxic effect against MDA-MB-231 and DLD
cell lines, respectively, with IC50 values of 25 and 19 lM.
Synthesised compounds did not have activity against MCF-
7 cancer cells. These peptides were nontoxic against pig’s
erythrocytes in vitro.
plasminogen into its active form plasmin, a broad spectrum
serine protease, which participates in the fibrinolytic cas-
cade (Collen 2001; Rijken and Sakharov 2001). uPA and
plasmin are strongly associated with tumor cells by influ-
encing tumor initiation, proliferation, migration, invasion,
metastasis and apoptosis (Andreasen et al. 1997; Duffy
2004; Mazar et al. 1999). Both these enzymes directly
promote proteolysis. However, plasmin acts also indirectly
through the activation of matrix metaloproteases. Overex-
pression of uPA has been found in various malignant
tumors, especially in the gastrointestinal, respiratory, gen-
itourinary systems, and in bones, bone marrow, skin, breast
and brain (Dass et al. 2008). High levels of urokinase are
correlated with enhanced tumor invasiveness, metastasis
and poor prognosis in certain cancers (Sidenius and Blasi
2003).
Keywords Urokinase inhibitor ꢀ Anti-amidolytic activity ꢀ
The inhibition of plasminogen activation by uPA
appears to be an attractive approach to cancer therapy,
especially against tumor growth and metastasis (Rockway
et al. 2002; Rockway 2003; Schweinitz et al. 2004). In
1997, Ke et al. analyzed a peptide library and selected the
primary peptide sequence SGRSA (from position P3 to P2)
as the substrate of uPA and tPA. Zeslawska et al. (2000,
2003) investigated the structure–function relationship of
certain peptides such as phenethyl-sufonamidino-D-seryl-L-
alanyl-L-argininal, and peptidomimetic inhibitors of uPA
(e.g., benzamidine, amiloride, 2,4,6-triisopropyl-phe-
nylsulfonyl-L-(3-amidino)-phenylalanine-piperazine-N-b-
alanine). Studies have demonstrated that the S1 pocket of
uPA is the critical site for interaction with inhibitors. The
P1 positively charged residue of an inhibitor (arginine,
benzamidine or phenylguanidine) makes a salt bridge
with Asp189 in the S1 pocket of uPA. The P2 residue of
the potential inhibitor, which should be hydrophobic, is
accommodated in S2 side. When the P3 position is
Low molecular weight peptides
Introduction
Urokinase plasminogen activator (uPA), a trypsin-like
serine protease, plays an important role in several biolog-
ical processes including tissue remodeling, cell migration
and matrix degradation (Irigoyen et al. 1999; Syrovets and
Simmet 2004). The primary role of uPA is converting
A. Markowska (&) ꢀ I. Bruzgo ꢀ K. Midura-Nowaczek
Department of Organic Chemistry, Medical University
of Bialystok, Bialystok, Poland
e-mail: agnieszka.markowska@umwb.edu.pl
W. Miltyk
Laboratory of Drug Analysis, Medical University of Bialystok,
Bialystok, Poland
123

48
Int J Pept Res Ther (2011) 17:47–52
occupied by an unnatural D-amino acid, the side-chain
projects into the S4 pocket. The residue of D-Ser could
form a favorable interaction with His-99 in the S4 site of
uPA.
Table 1 Amino acid insertion in novel peptides
Peptide
Inserted amino acid
1
2
3
4
5
6
7
8
9
10
a-Metyloalanine
a-Aminobutanoic acid
Valine
Recently, we described a series of peptide analogs
containing arginine as inhibitors of urokinase. The first
Norvaline
series
was
H(Ac)–D-Ser–Ala–(Gly)–Arg–OH(NH2)
Leucine
(Markowska et al. 2008) and the most active inhibitor of
urokinase was H–D-Ser–Gly–Arg–OH with an IC50 value
of 1 mM. The second series of peptide analogs of arginine
we synthesized were the peptides of the general formula
H–D-Ser–Ala–Arg–NH–X (Markowska et al. 2010), where
X = (CH2)n–NH2, n = 2–9, (CH2)m–OH, m = 2–4. H–D-
Ser–Ala–Arg–NH–(CH2)5–NH2 inhibited urokinase with a
Ki value of 6.3 lM. The Lineweaver–Burke analysis of the
H–D-Ser–Ala–Arg–NH–(CH2)5–NH2 compound proved
this compound to be a competitive inhibitor of urokinase.
The third series of compounds with D-Ser–Ala–Arg
sequence were N-sulfonylamides peptides (Markowska
et al. 2010). 2,4,6-triisopropylphenyl–SO2–D-Ser–Ala–
Arg–OH was the most selective inhibitor of urokinase and
a-tolyl–SO2–D-Ser–Ala–Arg–OH was the most active
inhibitor of uPA with a Ki value of 24 lM.
The purpose of this paper was the synthesis and the
amidolytic activity of 10 novel peptides against urokinase,
thrombin, trypsin, plasmin, tissue plasminogen activator
(t-PA) and kallikrein in vitro. We also tested the hemolytic
activity of the peptides against swine erythrocytes and the
anti-tumor activity against the following human breast
cancer cells: standard MCF-7, estrogen-independent MDA-
MB-231 and colorectal adenocarcinoma tumor DLD cell
line.
Norleucine
Izoleucine
tert-Leucine
Homoleucine
Neoglycine
the O-(benzotriazol-1-yl)-N,N,N0,N0-tetramethyluronium tet-
rafluoroborate, DIC = diisopropylcarbodiimide, NMP =
1-methyl-2-pirrolidon, Fmoc–L-Leu–OH, Fmoc–L-NLe–OH,
Fmoc–L-Ile–OH, Fmoc–L-Val–OH, Fmoc–L-NVa–OH,
Fmoc–Aib–OH, Fmoc–L-2Abu–OH, Fmoc–L-HLeu–OH,
Fmoc–L-TLe–OH, Fmoc–L-NptGly–OH were obtained from
Iris Biotech GmbH (Marktrewitz, Germany). DCM =
dichloromethane and DMF = dimethylformamide were the
products of Chempur (Piekary Slaskie, Poland) DCM was
used without further purification. DMF was distillated over
ninhidrin and stored under molecular sieves 4A. HPLC
solvent acetonitrile was purchased from Merck (Darmstadt,
Germany). Urokinase, trypsin, kallikrein, amiloride and
Bzl–L-Arg–pNAꢀHCl (Bzl = benzyl) were purchased from
Sigma (Schnelldorf, Germany). Plasmin, S-2444 (pyro-Glu–
Gly–Arg–pNAꢀHCl), S-2238 (H–D-Phe–Pip–Arg–pNA),
S-2251 (H–D-Val–Leu–Lys–pNA), S-2266 (H–D-Val–Leu–
Arg–pNAꢀ2HCl and S-2288 (H–D-Ile–Pro–Arg–pNA) were
obtained from Chromogenix (Milano, Italy). Thrombin and
phosphate buffered saline (PBS) were purchased from
Materials and Methods
´
Lubelska Wytwornia Szczepionek (Lublin, Poland). t-PA
was obtained from Boehringer Ingelheim GmbH (Ingelheim,
Germany).
The inserted amino acid (AA) in each of the novel peptides
was either leucine, norleucine, izoleucine, valine, norval-
ine, a-metyloalanine, a-aminobutanoic acid, homoleucine,
tert-leucine or neoglycine. Table 1 represents the identity
of the inserted amino acid in each of the 10 peptides syn-
thesized from X–D-Ser–AA–Arg–OH.
Peptide Synthesis
The peptides shown in Table 1 were synthesized manually
using the standard Fmoc-based strategy (Chan and White
2000). Fmoc deprotection steps were carried out with 20%
(v/v) piperidine in DMF/NMP (1:1) for 15 min. The cou-
pling reactions of Fmoc amino acids were performed in
DMF/NMP/DCM (1:1:1) using a molar ratio of amino acid/
DIC/HOBt/resin 3:3:3:1. In the case of the coupling of
Fmoc–D-Ser(t-Bu)–OH molar ratio of amino acid/TBTU/
HOBt/DIPEA/resin was 2:2:2:4:1. The reactions were
monitored with the Steward chloranil test. The cleavage
from the resin was carried out with TFA/water (95/5). After
2.5 h stirring, the resin was filtered and washed with TFA.
Source of Reagents
Fmoc–Arg(Pbf)–OH (Fmoc = 9-fluorenylmethyloxycarb-
onyl, Pbf = pentamethyl-dihydrobenzofuran), chloranil,
acetaldehyde, HOBt = 1-hydroxybenzotriazole were pur-
chased from Fluka (Schnelldorf, Germany). Fmoc–D-Ser
(t-Bu)–OH (t-Bu = t-butyl) and 2-chlorotrityl chloride
resin were purchased from Merck (Novabiochem, Darmstadt,
Germany). TFA = trifluoroacetic acid, DIPEA = diisopro-
pylethylamine, piperidine, TBTU = tetrafluoroborate salt of
123

Int J Pept Res Ther (2011) 17:47–52
49
d. borane buffer: 0.5 cm3 (pH 7.5), enzyme: trypsin (0.4
units/cm3), synthetic substrate: Bzl–L-Arg–pNAꢀHCl
(0.2 cm3, 8 mM/dm3);
e. Tris buffer: 0.6 cm3 (pH 9.0), enzyme: kallikrein
(3 units/cm3), synthetic substrate: S-2266 (0.1 cm3,
7.5 mM/dm3);
f. Tris buffer: 0.6 cm3 (pH 8.4), enzyme: t-PA (1.67
mg/cm3), synthetic substrate: S-2288 (0.1 cm3, 10 mM/
dm3).
The combined filtrates were concentrated under reduced
pressure. The crude peptide was washed with cold diethyl
ether, filtered, dissolved in glacial acetic acid and
lyophilized.
The Shimadzu LC-10A system (Smith and Hanly 1997;
Hartmann et al. 2009) (Shimadzu Europa GmbH, Duis-
burg, Germany) was used for analytical and semiprepara-
tory HPLC (Phenomenex C18, Jupiter 90A, 4 lm,
250 9 10 mm; Phenomenex C18, Jupiter 300A, 5 lm,
250 9 4 mm; solvents: A, 0.1% aqueous TFA; B, 0.1%
TFA in acetonitrile, gradient 0% B to 100% B in A in
30 min, flow rate 1 ml/min, monitored at 220 nm). The
major peak fraction was pooled and lyophilized. The
molecular weight determination was performed by mass
spectrometry using a Bruker Daltonics Esquire 6000
(Bruker Daltonik GmbH, Leipzig, Germany) with electro-
spray ionization (ESI), (Table 2).
The mixture was incubated for 3 min at 37°C, followed
by the addition of synthetic substrate solution in the same
buffer. After 20 min of incubation, the reaction was stop-
ped by adding 0.1 cm3 of 50% acetic acid and the absor-
bance of the released p-nitroaniline was measured at
405 nm. As shown in Table 3, each value represents the
average of a triplicate determination. The IC50 value for
each peptide was considered as the concentration of the
inhibitor, which decreased the absorbance at 405 nm by
50%, compared with the absorbance measured under the
same conditions without the inhibitor. Also, Ki was cal-
culated for each peptide from IC50 based on Cheng–Prusoff
equation (Cheng and Prusoff 1973). The results are shown
in Table 3.
Enzymatic Investigations
Determination of amidolytic activity was performed as
previously described (Okada et al. 1988). A detailed
description of the method is given below. Buffer and
0.1 cm3 of enzyme solution was added to 0.2 cm3 of
examined compound (1–10) (as control 0.15 M NaCl). The
buffer and the enzyme solution included the following
constituents:
Our results were compared with the data obtained by
Tamura et al. (2000) for 2-phenethyl–SO2–D-Ser–Ala–
Arg–al, the irreversible urokinase inhibitor with the same
tripeptide sequence. The determination methods were
identical.
a. Tris buffer: 0.6 cm3 (pH 8.8), enzyme: urokinase (50
units/cm3), synthetic substrate: S-2444 (0.1 cm3,
3 mM/dm3);
Tissue Culture
b. Tris buffer: 0.5 cm3 (pH 8.4), enzyme: thrombin
(1 units/cm3), synthetic substrate: S-2238 (0.2 cm3,
0.75 mM/dm3);
c. Tris buffer: 0.5 cm3 (pH 7.4), enzyme: plasmin (0.4
units/cm3), synthetic substrate: S-2251 (0.2 cm3,
3 mM/dm3);
All studies were performed on MCF-7, MDA-MB-231 and
DLD cells lines were purchased from American Type
Culture Collection, (Rockville, MD). The cells were
maintained in DMEM supplemented with 5% fetal bovine
serum (FBS), 2 mmol/ml glutamine, 50 U/ml penicillin,
50 mg/ml streptomycin at 37°C in a 5% CO2 incubator.
Table 2 Analytical data of the
synthesized peptides
Peptide no.
Yield (%)
Retention
time (min)
Melting
point (°C)
MW
[M?H]?
1
2
3
4
5
6
7
8
9
10
46
47
45
45
47
56
43
59
45
58
8.93
8.89
223–225
231–234
211–212
243–245
219–220
232–233
228–229
197–199
221–225
138–139
346.4
346.4
360.4
360.4
374.4
374.4
374.4
374.4
388.5
388.4
347.7
347.6
361.7
361.3
375.6
375.5
375.6
375.6
389.4
389.5
9.72
9.99
11.32
11.27
10.99
10.93
13.08
12.10
123

50
Int J Pept Res Ther (2011) 17:47–52
Table 3 Inhibition of amidolytic activity of enzymes against synthetic peptides with the general H–D-Ser–AA–Arg–OH formula
No. AA
Ki (lM)
Urokinase S-2444
Thrombin
S-2238
Trypsin Bzl–L-Arg–
pNAꢀHCl
Plasmin S-2251 t-PA S-2288
1.
2.
3.
4.
5.
6.
7.
8.
9.
a-Metyloalanine
n.i.
n.i.
n.i.
59.09 4.73
2.54 0.20
n.i.
n.i.
a-Aminobutanoic acid
Valine
n.i.
1.19 0.09 4.73 0.38
182.92 14.63
0.85 0.07
219.51 17.56
4.45 0.34
158.53 12.68
26.62 2.15
n.i.
n.i.
n.i.
4.48 0.36
n.i.
163.63 13.09 n.i.
Norvaline
4.36 0.35
27.27 2.18
40.9 3.27
1.81 0.15
31.81 2.55
n.i.
n.i.
Leucine
1.1 0.09 4.73 0.38
154.54 12.36
Norleucine
Izoleucine
n.i.
n.i.
n.i.
n.i.
0.77 0.06
19.09 1.53
tert-Leucine
Homoleucine
1.43 0.11 n.i.
n.i.
n.i.
n.i.
3.74 0.30
n.i.
10. Neoglycine
n.i.
n.i.
–
n.i.
163.63 13.09
–
2-Ph–(CH2)2–SO2–D-Ser–Ala–
Arg–al
3 nM (Tamura et al. 2000) –
–
Benzyl–SO2–D-Ser–4-
amidinobenzylamid
7.7 nM (Zeslawska et al.
2003)
–
–
–
–
Amiloride
7 lM (Zeslawska et al.
2003)
n.i. No inhibition was observed at maximum concentration (0.02 M)
Cytotoxicity Assay
Table 4 The nonviability of DLD and MDA-MB-231 cells treated
for 24 h with different concentrations of the synthesized inhibitors
The toxicity of the evaluated compounds was determined by
the method of Plumb et al. (Plumb et al. 1989). MCF-7,
MDA-MB-231 and DLD cells were maintained as descri-
bed above. After 48 h of incubation of the cells with syn-
thesized peptides, the medium was discarded and the cells
were rinsed three times with PBS. The cells were then incu-
bated for 4 h in 2 ml of PBS with 50 ml of MTT (5 mg/ml).
Afterremovalofthemedium, thecellswerelysedin200 mlof
DMSO with 20 ml of Sorensen’s buffer (0.1 M glycine with
0.1 M NaCl, pH 10.5). The absorbance was measured at
570 nm. The cytotoxic activity towards MDA-MB-231 and
DLD of synthesized peptides was calculated as percentage of
nonviable cells and the IC50 value was estimated from loga-
rithm curves as shown in Table 4.
Compound no.
IC50 (lM) (% of control 2)
DLD
MDA-MB-231
1
32
54
2
43
76
3
55
48
4
19
25
5
56
119
84
6
52
7
59
80
8
57
81
9
n.e.
n.e.
62.1
n.e.
n.e.
126
10
Amiloride
n.e. No cytotoxic effect was observed in maximum concentration
(250 lM)
Hemolytic Activity
a
Mean values SD from three independent experiments done in
duplicate
Pig’s fresh red blood cells (p-RBC) were washed three
times with PBS (35 M phosphate buffer/0.15 mM NaCl,
pH 7.4) and were centrifuged at 10009g for 10 min to
remove plasma and the buffy coat. Erythrocytes in sus-
pension were incubated with various concentrations of
peptides for 1 h at 37°C. The final erythrocyte concentra-
tion was 5% v/v. After the centrifugation (10009g for
10 min), 100 ll of the supernatant was transferred into
sterilized 96-well plates, where hemoglobin release was
monitored, using Infinite M200 plate reader and measuring
the absorbance at 414 nm. Zero hemolysis (blank), hemo-
lysis with amiloride as reference compound for synthesised
peptides and 100% hemolysis, which consisted of p-RBC
suspended in PBS and 0.1% Triton-X-100, were deter-
mined respectively. The percentage of hemolysis was
calculated with the following formula: %hemolysis =
(Abs414nm in the peptide solution in PBS/Abs414nm in 0.1%
Triton-X-100 in PBS) 9 100.
123

Int J Pept Res Ther (2011) 17:47–52
51
Results and Discussion
uPA is closely related to cell-surface events when incu-
bated with the breast cancer cells. MCF-7 had low uPAR/
uPA-expressing and low plasminogen-binding, whereas
MDA-MB-231 had high uPAR/uPA expressing and high
plasminogen-binding (Stillfried et al. 2007). Thus, the
influence of the synthetic peptides on the cytotoxic effect
of MCF-7 cells could be insignificant.
We obtained ten new peptides as potential inhibitors of
urokinase by the manual solid phase synthesis and tested
them for the effect on the amidolytic activities of uroki-
nase, thrombin, trypsin, plasmin, t-PA and kallikrein.
These peptides did not influence the enzymatic activity of
kallikrein.
We hope that our studies provided information about the
structure–activity and may be used for synthesizing more
active enzyme inhibitors and potential antitumor agents.
The results indicated that the concentration up to 1000
lg/ml of the synthesized peptides did not lyse erythrocytes.
The synthesized peptides were not selective inhibitors of
urokinase. Peptides 1 (H–D-Ser–Aib–Arg–OH) and 2 (H–D-
Ser–Abu–Arg–OH), containing two carbon atoms in their
amino acid side chains inhibited the activity of plasmin.
Peptides with three and four carbon atoms in the amino acid
side chains (H–D-Ser–Val–Arg–OH 3, H–D-Ser–NVal–Arg–
OH 4, H–D-Ser–Leu–Arg–OH 5, H–D-Ser–NLeu–Arg–OH
6, H–D-Ser–ILeu–Arg–OH 7, H–D-Ser–tLeu–Arg–OH 8)
inhibited plasmin and urokinase. Tripeptides 9 and 10,
containing homoleucine and neoglycine respectively as P2
residue, were non active inhibitors of plasmin and urokinase.
The most active peptide against urokinase was H–D-Ser–
NVal–Arg–OH 4 with a Ki value 0.85 lM.
Conclusion
Our investigation of the structure–activity relationship of a
series of new peptide sequences as potential urokinase
inhibitors reveals that the obtained H–D-Ser–NVal–Arg–
OH was the most active inhibitor of uPA with Ki 0.85 lM
value. The compound showed cytotoxic effects against
MDA-MB-231 cell lines with a IC50 25 lM value and with
a IC50 19 lM value against DLD cell lines. The examined
compound did not influence MCF-7 cancer cells.
The obtained values of Ki were higher than Ki of the
standard inhibitors. However, 2-phenethyl–SO2–D-Ser–
Ala–Arg–H was an alkylating agent and irreversibly
inhibited urokinase by forming a covalent adduct with an
active site of the enzyme (Zeslawska et al. 2003; Tamura
et al. 2000). Benzyl–SO2–D-Ser–4-amidinobenzylamid
with Ki = 0.0077 lM inhibitory activity was a non-pep-
tidic inhibitor of urokinase. 4-Amidinobenzylamine was a
decarboxylated arginine mimetic, which has been widely
used for the development of urokinase inhibitors (Rockway
2003; Schweinitz et al. 2004). The previous analysis of the
effect of H–D-Ser–Ala–Arg–NH–(CH2)5–NH2 on the
activity of urokinase showed that the derivatives with these
kinds of sequence reversibly and competitively inhibited
urokinase.
References
ˇ
Andreasen PA, Kjrller L, Christensen L, Duffy MJ (1997) The
urokinase-type plasminogen activator system in cancer metasta-
sis: a review. Int J Cancer 72:1–22
Chan WC, White PD (2000) Fmoc solid phase peptide synthesis.
Oxford University Press, New York
Cheng Y, Prusoff WH (1973) Relationship between the inhibition
constant (Ki) and the concentration of inhibitor, which causes 50
per cent inhibition (IC50) of an enzymatic reaction. Biochem
Pharmacol 22:3099–3108
Collen D (2001) Ham-Wasserman lecture: role of the plasminogen
system in fibrin-homeostasis and tissue remodeling. Hematology
1:1–9
Dass K, Ahmad A, Azmi AS, Sarkar SH, Sarkar FH (2008) Evolving
role of uPA/uPAR system in human cancers. Cancer Treat Res
34:122–136
The peptides 1–10 were tested in vitro for their antitu-
mour activity in human breast cancer cells: standard MCF-
7, estrogen-independent MDA-MB-231 and colorectal
adenocarcinoma tumor DLD line. Peptides 9 and 10 were
not active in vivo. The most interesting peptide was com-
pound 4 with an IC50 values of 19 lM for DLD cells and
25 lM for MDA-MB-231 cells, which was the most
selective inhibitor of urokinase. All potential inhibitors
were insignificantly less cytotoxic to MDA-MB-231 and
DLD cells than amiloride. Amiloride, which is a potas-
sium-sparing diuretic drug, has been shown to inhibit uPA
with Ki = 7 lM and to have antitumor activity in vivo
(Zeslawska et al. 2003).
Duffy MJ (2004) The urokinase plasminogen system: role in
malignancy. Curr Pharm Des 10:39–49
Hartmann BM, Kaar W, Yoo IK, Lua LHL, Falconer RJ, Middelberg
APJ (2009) The chromatography-free release, isolation and
purification of recombinant peptide for fibril self-assembly.
Biotechnol Bioeng 104:973–985
ˇ
´
Irigoyen JP, Munoz-Canoves P, Montero L, Koziczak M, Nagamine
Y (1999) The plasminogen activator system: biology and
regulation. Cell Mol Life Sci 56:104–132
Ke SH, Coombs GS, Tachias K, Corey DR, Madison EL (1997)
Optimal subsite occupancy and design of a selective inhibitor of
urokinase. J Biol Chem 272:20456–20462
Markowska A, Bruzgo I, Midura-Nowaczek K (2008) Effects of
tripeptides on the amidolytic activities of urokinase, thrombin,
plasmin and trypsin. Int J Pept Res Ther 14:215–218
Markowska A, Bruzgo I, Midura-Nowaczek K (2010a) Synthesis and
activity of amides of tripeptides as potential urokinase inhibitors.
J Enz Inh Med Chem 25:139–142
The synthesized peptides did not influence MCF-7
cancer cells. It was found that the proteolytic activity of
123

52
Int J Pept Res Ther (2011) 17:47–52
Markowska A, Bruzgo I, Midura-Nowaczek K (2010b) Synthesis and
activity of N-sulfonylamides of tripeptides as potential urokinase
inhibitors. Prot Pept Lett 17:1300–1304
Sidenius N, Blasi F (2003) The urokinase plasminogen activator
system in cancer: recent advances and implication for prognosis
and therapy. Cancer Metastasis Rev 22:205–222
Mazar AP, Henkin J, Goldfarb RH (1999) The urokinase plasminogen
activator system in cancer: implications for tumor angiogenesis
and metastasis. Angiogen 3:15–23
Smith DD, Hanly AM (1997) Purification of synthetic peptides by
high performance liquid chromatography. Neuropept Protoc
73:75–87
Okada Y, Tsuda Y, Teno N, Wanaka K, Bohgaki M, Hijikata-
Okunomiya A, Naito T, Okamoto S (1988) Synthesis of active
center-directed peptide inhibitors of plasmin. Chem Pharm Bull
36:1289–1297
Stillfried GE, Saunders DN, Ranson M (2007) Plasminogen binding
and activation at the breast cancer cell surface: the integral role
of urokinase activity. Breast Cancer Res 9:R14
Syrovets T, Simmet T (2004) Novel aspects and new roles for the
serine protease plasmin. Cel Mol Life Sci 61:873–885
Tamura S, Weinhouse MI, Roberts CA, Goldman EA, Masukawa K,
Anderson SM, Cohen CR, Bradbury AE, Bernardino VT, Dixon
SA, Ma MG, Nolan TG, Brunck TK (2000) Synthesis and
biological activity of peptidyl aldehyde urokinase inhibitors.
Biorg Med Chem Lett 10:983–987
Zeslawska E, Schweinitz A, Karcher A, Sondermann P, Sperl S,
Stu¨rzebecher J, Jacob U (2000) Crystals of the urokinase type
plasminogen activator variant beta(c)-uPA in complex with
small molecule inhibitors open the way towards structure-based
drug design. J Mol Biol 301:465–475
Plumb JA, Milroy R, Kaye SB (1989) Effects of the pH dependence
of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
mide-formazan absorption on chemosensitivity determined by
a novel tetrazolium-based assay. Cancer Res 49:4435–4440
Rijken DC, Sakharov DV (2001) Basic principles in thrombolysis:
regulatory role of plasminogen. Thromb Res 103:S41–S49
Rockway TW (2003) Small molecules inhibitors of urokinase-type
plasminogen activator. Expert Opin Ther Patents 13(6):773–786
Rockway TW, Nienaber V, Giranda VL (2002) Inhibitors of protease
domain of urokinase-type plasminogen activator. Curr Pharm
Design 8:2541–2558
Schweinitz A, Steimetzer T, Banke IJ, Arlt MJE, Stu¨rzebecher A,
Schuster O, Geissler A, Giersiefen H, Zesławska E, Jacob U,
Kr}uger A, St}urzebecher J (2004) Design of novel and selective
inhibitors of urokinase-type plasminogen activator with
improved pharmacokinetic properties for use as antimetastatic
agents. J Biol Chem 279:33613–33622
Zeslawska E, Jacob U, Schweinitz A, Coombs G, Bode W, Madison
EJ (2003) Crystals of urokinase type plasminogen activator
complexes reveal the binding mode of peptidomimetic inhibi-
tors. J Mol Biol 328:109–118
123