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

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 K_i value 5.02 μM and 5.7 μM, respectively.

Keywords::

• Urokinase inhibitor
• antiamidolytic activity
• low molecular peptide

Introduction

Serine proteases are enzymes responsible for vital processes in man such as digestion, blood coagulation, fibrinolysis, fertilization, apoptosis and immunity¹.

Plasmin is a serine protease that is central to a number 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-specificity^3–5. Most inhibitors of plasmin contain Phe-Lys(Arg) sequence^6–8.

Thrombin is responsible for the conversion of fibrinogen into fibrin, platelet activation and the feedback activation of other coagulation factors. The 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 preference to proline, it has not strong correlation at the P3 and has a preference for aliphatic amino acids in the P4. Thrombin inhibitors are classified as indirect inhibitors (heparin) and direct inhibitors (hirudin, bivalirudin, argatroban, dabigatran^9,10). Argatroban and dabigatran mimic D-Phe-Pro-Arg thrombin peptide substrate.

Trypsin is secreted by the pancreas and takes part in the digestion of food proteins. This 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 enzyme^11,12.

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 metalloproteases, 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 invasiveness, metastasis and poor prognosis. Compounds with D-Ser-AA-4-amidinobenzylamide sequence (C-terminal fragment mimics Arg) inhibit the activity of urokinase¹⁵.

Recently we described a series of peptide analogs containing arginine as inhibitors of urokinase. The first series was H(Ac)-D-Ser-Ala-(Gly)-Arg-OH(NH₂)¹⁶. The 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. The 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 K_i value of 6.3 μM¹⁷. 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 peptides¹⁸. Methyl-SO₂-D-Ser-Ala-Arg-OH was the most selective inhibitor of trypsin with a K_i value of 4 μM. The 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 K_i 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. There are potent and selective tripeptide arginine aldehydes as thrombin inhibitors with 1,2-disubstituted cyclohexane derivatives²⁰. Unnatural amino acid in potential peptide drugs limit conformational flexibility, enhance enzymatic stability and improve pharmacodynamics and bioavailability. We present the synthesis of peptides of the general X-D-Ser-AA-Arg-OH formula where AA = phenylglycine, phenylalanine, homophenylalanine, cyclohexylglycine, cyclohexylalanine, homocyclohexylalanine, 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 kallikrein of the synthesized peptides. 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.

Table 1.  Structure of obtained peptides: H-D-Ser-AA-Arg-OH.

Compound	AA
1	Phenylglycine	Phg
2	Phenylalanine	Pha
3	Homophenylalanine	homoPhe
4	Cyclohexylglycine	Chg
5	Cyclohexylalanine	Cha
6	Homocyclohexylalanine	homoCha
7	α-methylphenylalanine	α-MePhe
8	1-Aminocyclohexyl carboxylic acid	1-ACH

Materials and methods

Reagents

Fmoc-Arg(Pbf)-OH (Fmoc = 9-fluorenylmethyloxycarbonyl, Pbf = penta-methyldihydrobenzofuran), chloranil, acetaldehyde, HOBt = 1-hydroxybenzotriazole 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 = diisopropylethylamine, 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-formamide were the products of Chempur (Piekary Slaskie, Poland). DCM was used without further purification. 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). Thrombin 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

The peptides shown in Table 1 were synthesized manually using the standard Fmoc-based strategy²¹. Fmoc deprotection steps were carried out with 20% (v/v) piperidine in DMF/NMP (1:1) for 15 min. The 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 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. The combined filtrates were concentrated under reduced pressure. The crude peptide was washed with cold diethyl ether, filtered, dissolved in water and lyophilized.

The 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 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 electrospray ionization (ESI), (Table 2).

Table 2.  Analytical data of the synthesized compounds.

No	Yield [%]	Retention time [min.]	MW	[M+H]^+
1	57	12.87	408.4	409.6
2	46	11.97	394.4	395.5
3	51	10.46	422.5	423.4
4	52	13.56	414.5	415.7
5	49	14.48	400.5	401.3
6	50	15.33	428.5	429.6
7	48	11.67	422.5	423.7
8	49	12.51	386.5	387.4

Enzymatic investigations

Determination of amidolytic activity was performed as previously described²². The 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 NaCl^1–8 (as control 0.15 M NaCl). The buffer and the enzyme solution included:

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 (1.67 mg/mL), synthetic substrate: S-2288 (0.1 mL, 10 mM).

The 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 triplicate determination. IC₅₀ value 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. K_i was calculated from IC₅₀ based on Cheng–Prusoff equation²³. The results are given in Table 3.

Table 3.  Inhibition of H-D-Ser-AA-Arg-OH on the amidolytic activity of enzymes.

No	AA	Ki [µM]
		Urokinase S-2444	Thrombin S-2238	Trypsin Bzl-L-Arg-pNA HCl	Plasmin S-2251
1	Phg	487.8 ± 39.0	22.9 ± 1.8	26.3 ± 1.9	5.7 ± 0.5
2	Phe	544 ± 43.5	12.1 ± 0.9	22.9 ± 1.7	8.7 ± 0.7
3	homoPhe	565.8 ± 43.9	16.1 ± 1.3	23.9 ± 1.8	5.9 ± 0.5
4	Chg	321.9 ± 26.8	5.02 ± 0.4	48.4 ± 3.7	7.7 ± 0.7
5	Cha	246.97 ± 19.7	19.8 ± 1.6	30.9 ± 2.5	8.9 ± 0.7
6	homoCha	451.2 ± 36.6	18.1 ± 1.4	27.2 ± 1.9	11.2 ± 0.9
7	αMePhe	275.6 ± 21.9	12.47 ± 0.9	28.2 ± 2.2	7.9 ± 0.7
8	1ACH	395.1 ± 31.7	14.99 ± 1.2	26.2 ± 1.8	7.7 ± 0.7
	Ac-Leu-Leu-Arg-H	–	–	8 ± 0.7 nM	8.2 ± 0.7 nM
	Ac-Leu-Leu-Arg-COCHO	–	–	1.9 µM^Citation27	4.9 µM^Citation27

Our results were compared with the data obtained for Ac-Leu-Leu-Arg-H (leupeptin–natural, microbial origin, inhibitor of proteinases)²⁴.

Tissue culture

All studies were performed on MCF-7 and MDA-MB-231 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.

Cytotoxicity assay

The 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). The cells were then incubated for 4 h in 2 mL of PBS with 50 mL of MTT (5 mg/mL). After removal of the medium, the cells were lysed in 200 mL of 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 of synthesized peptides was calculated as percentage of nonviable cells and the IC₅₀ value was estimated from logarithm curves as shown in Table 4.

Table 4.  The nonviability of MDA cells treated for 24 h with different concentrations of the synthesized peptides.

Compounds	IC50 [μM] (% of control ± 2)a
1	306
2	283
3	272
4	245
5	263
6	270
7	278
8	258
Ac-Leu-Leu-Arg-H	580

^a Mean values ± SD from three independent experiments done in duplicate.

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. The various 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 erythrocyte concentration was 5% v/v). After the centrifugation (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 synthesized peptides and 100% hemolysis which consisted of p-RBC suspended in PBS and 0.1% Triton-X-100 were determined respectively. The percentage of hemolysis was calculated with the following formula:

Results and discussion

We obtained eight new compounds as potential inhibitors of urokinase by the manual solid phase synthesis. The examined compounds did not influence the enzymatic activity of kallikrein and t-PA. None of the compounds showed selectivity towards urokinase, plasmin, trypsin and thrombin. The results indicated that the concentration up to 1000 µg/mL of the synthesized peptides did not lyse erythrocytes. The synthesized peptides were not active inhibitors of urokinase, but active inhibitors of plasmin.

A lot of peptides were earlier reported as substrates of plasmin^26,27. The predicted P1 residue was arginine of the most of examined substrate sequence and lysine in remaining. The preference was selected for large aromatic 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 1 and 4 (H-D-Ser-Phg-Arg-OH and H-D-Ser-Chg-Arg-OH) had comparable K_i values of plasmin inhibition. The direct connection of the cyclic system (H-D-Ser-1-ACH-Arg-OH 8) with the 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). The substitution of α-hydrogen for methyl group in H-D-Ser-αMePhe-Arg-OH 7 has similar values of plasmin inhibition.

The obtained values of K_i were higher than K_i of the standard inhibitors. However, Ac-Leu-Leu-Arg-H²⁴, a natural inhibitor of serine proteases irreversibly inhibited 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.

The 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 binding²⁹. Due to synthesized peptides would be rather nontoxic to MCF-7 cancer cells. The peptides 1-8 were slightly less cytotoxic to MDA-MB-231 in comparison to the earlier described leupeptin.

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.

Declaration of interest

The authors declare no conflicts of interest.