Tripeptides with non-code amino acids as potential serine proteases inhibitors

Article abstract of DOI:10.3109/14756366.2011.651463

Eight peptides of the general H-D-Ser-AA-Arg-OH formula, where AA=phenylglycine, phenylalanine, homophenylalanine, cyclohexylglycine, cyclohexylalanine, homocyclohexylalanine, α-methylphenylalanine and 1-aminocyclohexyl carboxylic acid were obtained and t

Full text of DOI:10.3109/14756366.2011.651463

Journal of Enzyme Inhibition and Medicinal Chemistry, 2013; 28(3): 639–643
© 2013 Informa UK, Ltd.
ISSN 1475-6366 print/ISSN 1475-6374 online
DOI: 10.3109/14756366.2011.651463
SHORT COMMUNICATION
Tripeptides with non-code amino acids as potential serine
proteases inhibitors
Agnieszka Markowska1, Magdalena Bruzgo2, Arkadiusz Surażyński3, and Krystyna
Midura-Nowaczek1
1Department of Organic Chemistry, 2Department of Instrumental Analysis and 3Department of Medicinal Chemistry,
Medical University of Bialystok, Poland
Abstract
Eight peptides of the general H-D-Ser-AA-Arg-OH formula, where AA=phenylglycine, phenylalanine,
homophenylalanine, cyclohexylglycine, cyclohexylalanine, homocyclohexylalanine, α-methylphenylalanine and
1-aminocyclohexyl carboxylic acid were obtained and tested for their effect on the amidolytic activities of urokinase,
thrombin, trypsin, plasmin, t-PA and kallikrein. We tested the hemolytic activity of the peptides against porcine
erythrocytes and the antitumor activity against the human breast cancer cells, standard MCF-7 and estrogen-
independent MDA-MB-231. The most active compounds were H-D-Ser-Chg-Arg-OH towards thrombin and H-D-Ser-
Phg-Arg-OH towards plasmin with Ki value 5.02 μM and 5.7 μM, respectively.
Keywords: Urokinase inhibitor, antiamidolytic activity, low molecular peptide
Introduction
argatroban, dabigatran9,10). Argatroban and dabigatran
Serine proteases are enzymes responsible for vital pro-
cesses in man such as digestion, blood coagulation,
fibrinolysis, fertilization, apoptosis and immunity1.
Plasmin is a serine protease that is central to a num-
ber of physiological processes such as lysis of fibrin clots,
tissue remodelling and cell migration2. It is also involved
in the pathological processes such as angiogenesis and
metastasis in cancer. Plasmin has substrate preference
for lysine in P1, for aromatic amino acids in P2 and broad
P3-specificity3–5. Most inhibitors of plasmin contain Phe-
mimic D-Phe-Pro-Arg thrombin peptide substrate.
Trypsin is secreted by the pancreas and takes part in
the digestion of food proteins. is enzyme is responsible
for cleaving peptide bonds following a positively-charged
amino acid residue similarly to plasmin and thrombin.
An aspartic acid residue in position 189 at the base of
the pocket is found in trypsin and it can interact with
positively-charged residues such as arginine and lysine
in substrates or inhibitors of the enzyme11,12
.
Urokinase (uPA), in contrast to plasmin, trypsin and
thrombin, is a high-specific trypsin-like protease13.
Urokinase activates plasminogen to plasmin which
can lead, through the activation of many matrix met-
alloproteases, to the increase in tissue destruction.
Overexpression of uPA has been found in various
malignant tumours, especially in the digestive system,
the respiratory system, bones, skin, breast, the genital
system, the urinary system, brain and leukemia14. High
levels of urokinase are correlated with enhanced inva-
siveness, metastasis and poor prognosis. Compounds
Lys(Arg) sequence6–8
.
rombin is responsible for the conversion of fibrino-
gen into fibrin, platelet activation and the feedback
activation of other coagulation factors. e substrate
specificity preference of thrombin is cleavage after P1
arginine over lysine and shows little difference in the
extended subsites5. At the P2 thrombin has strict pref-
erence to proline, it has not strong correlation at the P3
and has a preference for aliphatic amino acids in the P4.
rombin inhibitors are classified as indirect inhibitors
(heparin) and direct inhibitors (hirudin, bivalirudin,
with
D-Ser-AA-4-amidinobenzylamide
sequence
Address for Correspondence: Agnieszka Markowska, Department of Organic Chemistry, Medical University of Bialystok, Kilinski 1 Strt
15-089 Bialystok, Poland. E-mail: agnieszka.markowska@umb.edu.pl
(Received 23 September 2011; revised 15 December 2011; accepted 15 December 2011)
639

640 A. Markowska et al.
(C-terminal fragment mimics Arg) inhibit the activity
of urokinase15.
cancer cells, standard MCF-7 and estrogen-independent
MDA-MB-231.
Recently we described a series of peptide analogs
containing arginine as inhibitors of urokinase. e first
series was H(Ac)-D-Ser-Ala-(Gly)-Arg-OH(NH2)16. e
most active inhibitor of urokinase was H-D-Ser-Gly-
Arg-OH, and the most active and selective inhibitor of
plasmin was H-D-Ser-Gly-Arg-NH2, both with an IC50
value of 1 mM. e second series of peptide analogs of
arginine we synthesized were the peptides of the general
formula H-D-Ser-Ala-Arg-NH-X17, 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
μM17. e Lineweaver–Burke analysis of the H-D-Ser-
Ala-Arg-NH-(CH2)5-NH2 compound proved this com-
pound to be a competitive inhibitor of urokinase. e
third series of compounds with D-Ser-Ala-Arg sequence
were N-sulfonylamides peptides18. Methyl-SO2-D-Ser-
Ala-Arg-OH was the most selective inhibitor of trypsin
with a Ki value of 4 μM. e last group compounds we
published were peptides of the general H-D-Ser-AA-
Arg formula where AA were amino acids with aliphatic
side chains. H-D-Ser-NVal-Arg-OH was the most active
inhibitor of urokinase with Ki 0.85 µM value19. In this
series we obtained three active inhibitors of plasmin
with amino acids: α-aminobutanoic acid, tert-leucine
and leucine in P2.
On the basis of this peptide sequence we described
a series of tripeptides as inhibitors of serine trypsin-like
proteases with aromatic and/or cyclic amino acids in P2.
ere are potent and selective tripeptide arginine alde-
hydesasthrombininhibitorswith1,2-disubstitutedcyclo-
hexane derivatives20. Unnatural amino acid in potential
peptide drugs limit conformational flexibility, enhance
enzymatic stability and improve pharmacodynamics
and bioavailability. We present the synthesis of pep-
tides of the general X-D-Ser-AA-Arg-OH formula where
AA = phenylglycine, phenylalanine, homophenylalanine,
cyclohexylglycine, cyclohexylalanine, homocyclohexyla-
lanine, α-methylphenylalanine and 1-aminocyclohexyl
carboxylic acid (Table 1.). We also present the effect on
the amidolytic activities of urokinase, thrombin, trypsin,
plasmin, tissue-plasminogen activator (t-PA) and kal-
likrein of the synthesized peptides. We tested the hemo-
lytic activity of the peptides against porcine erythrocytes
and the antitumor activity against the human breast
Materials and methods
Reagents
Fmoc-Arg(Pbf)-OH
(Fmoc = 9-fluorenylmethyloxy-
carbonyl, Pbf = penta-methyldihydrobenzofuran),
chloranil, acetaldehyde, HOBt = 1-hydroxybenzo-
triazole were purchased 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 = diisopropyleth-
ylamine, DIC = diisopropylcarbodiimide, piperidine,
TBTU = tetrafluoroborate salt of the O-(benzotriazol-
1-yl)-N,N,N’,N’-tetramethyluronium tetrafluoroborate,
NMP = 1-methyl-2-pyrrolidon, Fmoc-L-Phg-OH, Fmoc-
L-Phe-OH, Fmoc-L-homoPhe-OH, Fmoc-L-Chg-OH,
Fmoc-L-Cha-OH,
Fmoc-homoCha-OH,
Fmoc-L-
αMePhe-OH and Fmoc-L-1-ACH-OH were obtained
from Iris Biotech GmbH (Marktrewitz, Germany).
DCM = dichloromethane and DMF = dimethyl-forma-
mide were the products of Chempur (Piekary Slaskie,
Poland). DCM was used without further purifica-
tion. DMF was distillated over ninhydrin and stored
under molecular sieves 4A. HPLC solvent acetonitrile
was purchased from Merck (Darmstadt, Germany).
Urokinase, trypsin, kallikrein and Bzl-L-Arg-pNA HCl
(Bzl = benzyl) were purchased from Sigma (Schnelldorf,
Germany). Plasmin, S-2444 (pyro-Glu-Gly-Arg-pNA
HCl), S-2238 (H-D-Phe-Pip-Arg-pNA), S-2251 (H-D-
Val-Leu-Lys-pNA), S-2266 (H-D-Val-Leu-Arg-pNA 2HCl
and S-2288 (H-D-Ile-Pro-Arg-pNA) were obtained from
Chromogenix (Milano, Italy). Ac-Leu-Leu-Arg-H was
purchased from Sigma (Poznan, Poland). rombin and
phosphate buffered saline (PBS) were purchased from
Lubelska Wytwórnia Szczepionek (Lublin, Poland).
t-PA was obtained from Boehringer Ingelheim GmbH
(Ingelheim, Germany).
Experimental
Peptide synthesis
e peptides shown in Table 1 were synthesized manu-
ally using the standard Fmoc-based strategy 21. Fmoc
deprotection steps were carried out with 20% (v/v)
piperidine in DMF/NMP (1:1) for 15 min. e coupling
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 cou-
pling of Fmoc-D-Ser(t-Bu)-OH molar ratio of amino
acid/TBTU/HOBt/DIPEA/resin was 2:2:2:4:1. e reac-
tions were monitored with the Steward chloranil test.
e 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. e combined filtrates were con-
centrated under reduced pressure. e crude peptide
Table 1. Structure of obtained peptides: H-D-Ser-AA-Arg-OH.
Compound
AA
1
2
3
4
5
6
7
8
Phenylglycine
Phg
Phenylalanine
Pha
Homophenylalanine
Cyclohexylglycine
Cyclohexylalanine
Homocyclohexylalanine
α-methylphenylalanine
1-Aminocyclohexyl carboxylic acid
homoPhe
Chg
Cha
homoCha
α-MePhe
1-ACH
Journal of Enzyme Inhibition and Medicinal Chemistry

Tripeptides with non-code amino acids as potential serine proteases inhibitors 641
was washed with cold diethyl ether, filtered, dissolved
in water and lyophilized.
triplicate determination. IC50 value was considered as
the concentration of the inhibitor, which decreased the
absorbance at 405 nm by 50%, compared with the absor-
bance measured under the same conditions without the
inhibitor. Ki was calculated from IC50 based on Cheng–
Prusoff equation23. e results are given in Table 3.
Our results were compared with the data obtained for
Ac-Leu-Leu-Arg-H (leupeptin–natural, microbial origin,
inhibitor of proteinases)24.
e Shimadzu LC-10A system (Shimadzu Europa
GmbH, Duisburg, Germany) was used for analytical and
semipreparatory HPLC (Phenomenex C18, Jupiter 90A,
4 µ, 250 × 10 mm; Phenomenex C18, Jupiter 300A, 5 µ,
250 × 4 mm; solvents: A, 0.1% aqueous TFA; B, 0.1% TFA
in acetonitrile, gradient 0% B to 100% B in A in 30min,
flow rate 1 ml/min, monitored at 220 nm). e major
peak fraction was pooled and lyophilized. e molecu-
lar weight determination was performed by mass spec-
trometry using a Bruker Daltonics Esquire 6000 (Bruker
Daltonik GmbH, Leipzig, Germany) with electrospray
ionization (ESI), (Table 2).
Tissue culture
All studies were performed on MCF-7 and MDA-MB-231
cells lines were purchased from American Type Culture
Collection, (Rockville, MD). e 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.
Enzymatic investigations
Determination of amidolytic activity was performed as
previously described22. e detailed description of the
method is given below. Buffer and 0.1 mL of enzyme
solution was added to 0.2 mL of examined compound
dissolved in 0.15 M NaCl1–8 (as control 0.15 M NaCl). e
buffer and the enzyme solution included:
Cytotoxicity assay
e toxicity of the evaluated peptides was determined
by the method of Plumb et al.25 in 10 μM, 100 μM, 250
μM, 500 μM and 1000 μM concentrations. MCF-7 and
MDA-MB-231 cells were maintained as described above.
After 48 h of incubation of the cells with synthesized
peptides, the medium was discarded and the cells were
rinsed three times with phosphate buffered saline (PBS).
e cells were then incubated for 4h in 2 mL of PBS with
50 mL of MTT (5 mg/mL). After removal of the medium,
the cells were lysed in 200mL of DMSO with 20mL of
Sorensen’s buffer (0.1 M glycine with 0.1 M NaCl, pH
10.5). e absorbance was measured at 570 nm. e
cytotoxic activity of synthesized peptides was calculated
as percentage of nonviable cells and the IC50 value was
estimated from logarithm curves as shown in Table 4.
a. Tris buffer − 0.6 mL (pH 8.8), enzyme: urokinase
(50 units/mL), synthetic substrate: S-2444 (0.1 mL,
3 mM);
b. Tris buffer − 0.5 mL (pH 8.4), enzyme: thrombin
(1 units/mL), synthetic substrate: S-2238 (0.2 mL,
0.75 mM);
c. Tris buffer − 0.5 mL (pH 7.4), enzyme: plasmin (0.4
units/mL), synthetic substrate: S-2251 (0.2 mL,
3 mM);
d. Borane buffer − 0.5 mL (pH 7.5), enzyme: trypsin (0.4
units/mL), synthetic substrate: Bzl-L-Arg-pNA HCl
(0.2 mL, 8 mM);
e. tris buffer − 0.6 ml (pH 9.0), enzyme: kallikrein (3
units/mL), synthetic substrate: S-2266 (0.1 mL,
7.5 mM);
f. Tris buffer − 0.6 ml (pH 8.4), enzyme: t-PA (1.67mg/
mL), synthetic substrate: S-2288 (0.1 mL, 10 mM).
Hemolytic activity
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 centrifugated at 1000g for 10 min
to remove plasma and the buffy coat. e various
e mixture was incubated for 3 min at 37°C. After 20 min
of incubation, the reaction was stopped by adding 0.1 ml
of 50% acetic acid, and the absorbance of the released
p-nitroaniline was measured at 405 nm (Spekol 1300,
AnalyticJena). Every value represents the average of the
Table 3. Inhibition of H-D-Ser-AA-Arg-OH on the amidolytic activity of
enzymes.
Ki [µM]
Trypsin
Urokinase
S-2444
rombin Bzl-L-Arg- Plasmin
No AA
S-2238
pNA HCl
26.3 1.9
22.9 1.7
23.9 1.8
48.4 3.7
30.9 2.5
S-2251
5.7 0.5
8.7 0.7
5.9 0.5
7.7 0.7
8.9 0.7
1
2
3
4
5
6
7
8
Phg
487.8 39.0
544 43.5
22.9 1.8
12.1 0.9
16.1 1.3
5.02 0.4
19.8 1.6
18.1 1.4
Phe
Table 2. Analytical data of the synthesized compounds.
[M+H]+
409.6
395.5
423.4
415.7
401.3
429.6
423.7
387.4
homoPhe
Chg
565.8 43.9
321.9 26.8
246.97 19.7
451.2 36.6
No
1
Yield [%] Retention time [min.]
MW
408.4
394.4
422.5
414.5
400.5
428.5
422.5
386.5
57
46
51
52
49
50
48
49
12.87
11.97
10.46
13.56
14.48
15.33
11.67
12.51
Cha
2
homoCha
αMePhe
1ACH
27.2 1.9 11.2 0.9
3
275.6 21.9 12.47 0.9
395.1 31.7 14.99 1.2
28.2 2.2
26.2 1.8
7.9 0.7
7.7 0.7
4
5
Ac-Leu-Leu-
Arg-H
–
–
8
0.7 nM 8.2 0.7 nM
6
7
Ac-Leu-Leu-
Arg-COCHO
–
–
1.9 µM27 4.9 µM27
8
© 2013 Informa UK, Ltd.

642 A. Markowska et al.
α-carbon does not change the activity of the examined
enzymes. Also elongation of methylene chain with
cyclic system connected to α-carbon does not change
the enzymes inhibition (H-D-Ser-homoPhe-Arg-OH 3
and H-D-Ser-homoCha-Arg-OH 6). e substitution
of α-hydrogen for methyl group in H-D-Ser-αMePhe-
Arg-OH 7 has similar values of plasmin inhibition.
e obtained values of Ki were higher than Ki of the
standard inhibitors. However, Ac-Leu-Leu-Arg-H24, a
natural inhibitor of serine proteases irreversibly inhib-
ited proteolytic enzymes by forming a hemi-acetal
covalent bond with an active site of the enzyme. But
despite the presence of extra carbonyl group in Ac-Leu-
Leu-Arg-COCHO28, which is a synthetic serine proteases
inhibitor, α-keto-β-aldehyde was comparably active
towards to synthesized peptide. Some peptides with
aliphatic side chain of amino acids in P2 previously
obtained, were more active towards urokinase, but only
3 of 10 (α-aminobutanoic acid, norvaline, isoleucine in
P2 in H-D-Ser-AA-Arg-OH) were more active plasmin
inhibitors19.
e synthesized peptides did not influence MCF-7
cancer cells. It was found that the proteolytic activity
of uPA is closely related to cell-surface events when
incubated 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 29. Due to synthesized
peptides would be rather nontoxic to MCF-7 cancer
cells. e peptides 1-8 were slightly less cytotoxic to
MDA-MB-231 in comparison to the earlier described
leupeptin.
Table 4. e nonviability of MDA cells treated for 24h with
different concentrations of the synthesized peptides.
Compounds
IC50 [μM] (% of control 2)a
1
306
283
272
245
263
270
278
258
580
2
3
4
5
6
7
8
Ac-Leu-Leu-Arg-H
aMean values SD from three independent experiments done in
duplicate.
concentrations of peptides (100 μg/mL, 250 μg/mL,
500 μg/mL and 1000 μg/mL) were incubated with the
erythrocyte suspension for 1 h at 37°C (the final eryth-
rocyte concentration was 5% v/v). After the centrifu-
gation (1000 g for 10 min), 100 µL of the supernatant
was transferred into sterilized 96-well plates, where
hemoglobin release was monitored with the use of the
Infinite M200 plate reader by measuring the absorbance
at 414 nm. Zero hemolysis (blank), hemolysis with
Ac-Leu-Leu-Arg-H as reference compound for synthe-
sized peptides and 100% hemolysis which consisted of
p-RBC suspended in PBS and 0.1% Triton-X-100 were
determined respectively. e percentage of hemolysis
was calculated with the following formula:






Abs414 nm in the peptide solution in PBS
Abs414 nm in 0.1% Triton-X-100 in PBS
% hemolysis =
× 100
We hope that our studies provide information about
the structure-activity and may be used for synthesizing
more active enzyme inhibitors and potential antitumor
agents.
Results and discussion
We obtained eight new compounds as potential inhibi-
tors of urokinase by the manual solid phase synthesis.
e examined compounds did not influence the enzy-
matic activity of kallikrein and t-PA. None of the com-
pounds showed selectivity towards urokinase, plasmin,
trypsin and thrombin. e results indicated that the con-
centration up to 1000 µg/mL of the synthesized peptides
did not lyse erythrocytes. e synthesized peptides were
not active inhibitors of urokinase, but active inhibitors of
plasmin.
Declaration of interest
e authors declare no conflicts of interest.
References
1. Nduwimana J, Guenet L, Dorval I, Blayau M, Le Gall JY, Le Treut A.
Proteases. Ann Biol Clin (Paris) 1995;53:251–264.
A lot of peptides were earlier reported as substrates
of plasmin26,27. e predicted P1 residue was arginine of
the most of examined substrate sequence and lysine in
remaining. e preference was selected for large aro-
matic residues in P23. Despite (Tyr)Phe-Arg(Lys) being
the most common sequence in literature active towards
plasmin, we proved that not only the aromatic system but
also the cyclic system is necessary to inhibit the activity
of plasmin. Peptide 2 and 5 (H-D-Ser-Phe-Arg-OH and
H-D-Ser-Cha-Arg-OH) or peptide 1and 4(H-D-Ser-Phg-
Arg-OH and H-D-Ser-Chg-Arg-OH) had comparable Ki
values of plasmin inhibition. e direct connection of
the cyclic system (H-D-Ser-1-ACH-Arg-OH 8) with the
2. Longstaff C, elwell C. Understanding the enzymology of
fibrinolysis and improving thrombolytic therapy. FEBS Lett
2005;579:3303–3309.
3. Hervio LS, Coombs GS, Bergstrom RC, Trivedi K, Corey DR,
Madison EL. Negative selectivity and the evolution of protease
cascades: the specificity of plasmin for peptide and protein
substrates. Chem Biol 2000;7:443–453.
4. Gechtman Z, Sharma R, Kreizman T, Fridkin M, Shaltiel S. Synthetic
peptides derived from the sequence around the plasmin cleavage
site in vitronectin. Use in mapping the PAI-1 binding site. FEBS
Lett 1993;315:293–297.
5. Backes BJ, Harris JL, Leonetti F, Craik CS, Ellman JA. Synthesis of
positional-scanning libraries of fluorogenic peptide substrates to
define the extended substrate specificity of plasmin and thrombin.
Nat Biotechnol 2000;18:187–193.
Journal of Enzyme Inhibition and Medicinal Chemistry

Tripeptides with non-code amino acids as potential serine proteases inhibitors 643
6. Tsuda Y, Teno N, Okada Y, Wanaka K, Bohgaki M, Hijikata-
19. MarkowskaA, BruzgoI, MiltykW, Midura-NowaczekK. Tripeptides
with C-Terminal Arginine as Potential Inhibitors of Urokinase. Int J
Pept Res er 2011;17:47–52.
20. Harmat NJ, Di Bugno C, Criscuoli M, Giorgi R, Lippi A, Martinelli A
et al. 1,2-disubstituted cyclohexane derived tripeptide aldehydes
as novel selective thrombin inhibitors. Bioorg Med Chem Lett
1998;8:1249–1254.
Okunomiya A et al. Synthesis of tripeptide chloromethyl ketones
and examination of their inhibitory effects on plasmin and plasma
kallikrein. Chem Pharm Bull 1989;37:3108–3111.
7. Markowska A, Bruzgo I, Midura-Nowaczek K. Methylketone
inhibitors of plasmin. Pharmazie 2006;61:898–900.
8. MarkowskaA,Midura-NowaczekK,BruzgoI.Lowmolecularpeptides
as potential inhibitors of plasmin. Acta Pol Pharm 2007;64:355–358.
9. Weitz JI. A novel approach to thrombin inhibition. romb Res
2003;109:S17–S22.
10. Altenburger JM, Lassalle GY, Matrougui M, Galtier D, Jetha JC,
Bocskei Z et al. SSR182289A, a selective and potent orally active
thrombin inhibitor. Bioorg Med Chem 2004;12:1713–1730.
11. OlsenJV,OngSE,MannM.TrypsincleavesexclusivelyC-terminalto
arginine and lysine residues. Mol Cell Proteomics 2004;3:608–614.
12. Navaneetham D, Sinha D, Walsh PN. Mechanisms and specificity
of factor XIa and trypsin inhibition by protease nexin 2 and basic
pancreatic trypsin inhibitor. J Biochem 2010;148:467–479.
13. Ke SH, Coombs GS, Tachias K, Corey DR, Madison EL. Optimal
subsite occupancy and design of a selective inhibitor of urokinase.
J Biol Chem 1997;272:20456–20462.
21. Chan WC, White PD. Fmoc Solid Phase Peptide Synthesis. Oxford
University Press: New York 2000.
22. Okada Y, Tsuda Y, Teno N, Wanaka K, Bohgaki M, Hijikata-
Okunomiya A et al. Synthesis of active center-directed peptide
inhibitors of plasmin. Chem Pharm Bull 1988;36:1289–1297.
23. Cheng Y, Prusoff WH. Relationship between the inhibition constant
(K1) and the concentration of inhibitor which causes 50 per cent
inhibition (I50) of an enzymatic reaction. Biochem Pharmacol
1973;22:3099–3108.
24. Aoyagi T, Miyata S, Nanbo M, Kojima F, Matsuzaki M. Biological
activities of leupeptins. J Antibiot 1969;22:558–568.
25. Plumb JA, Milroy R, Kaye SB. Effects of the pH dependence
of
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium
absorption on chemosensitivity
bromide-formazan
14. Duffy MJ. e urokinase plasminogen activator system: role in
malignancy. Curr Pharm Des 2004;10:39–49.
determined by a novel tetrazolium-based assay. Cancer Res
1989;49:4435–4440.
15. Schweinitz A, Steinmetzer T, Banke IJ, Arlt MJ, Stürzebecher
A, Schuster O et al. Design of novel and selective inhibitors
of urokinase-type plasminogen activator with improved
pharmacokinetic properties for use as antimetastatic agents. J Biol
Chem 2004;279:33613–33622.
16. Markowska A, Bruzgo I, Midura-Nowaczek K. Effects of tripeptides
on the amidolytic activities of urokinase thrombin, plasmin and
trypsin. Int J Pept Res er 2008;14:215–218.
26. Tsuda Y, Tada M, Wanaka K, Okamoto U, Hijikata-Okunomiya
A, Okamoto S et al. Structure-inhibitory activity relationship
of plasmin and plasma kallikrein inhibitors. Chem Pharm Bull
2001;49:1457–1463.
27. Enomoto R, Sugahara C, Komai T, Suzuki C, Kinoshita N, Hosoda A
et al. e structure-activity relationship of various YO compounds,
novel plasmin inhibitors, in the apoptosis induction. Biochim
Biophys Acta 2004;1674:291–298.
17. Markowska A, Bruzgo I, Midura-Nowaczek K. Synthesis and
activity of amides of tripeptides as potential urokinase inhibitors. J
Enzyme Inhib Med Chem 2010;25:139–142.
28. Lynas JF, Martin SL, Walker B. Synthesis and kinetic evaluation of
peptide α-keto-β-aldehyde-based inhibitors of trypsin-like serine
proteases. J Pharm Pharmacol 2001;53:473–480.
18. Markowska A, Bruzgo I, Miltyk W, Midura-Nowaczek K. Synthesis
and activity of N-sulfonylamides of tripeptides as potential
urokinase inhibitors. Protein Pept Lett 2010;17:1300–1304.
29. Stillfried GE, Saunders DN, Ranson M. Plasminogen binding and
activation at the breast cancer cell surface: the integral role of
urokinase activity. Breast Cancer Res 2007;9:R14.
© 2013 Informa UK, Ltd.