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Keywords:

  • proteinase inhibitors;
  • SFTI-1;
  • fluorescence

ABSTRACT

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8.  
  9. REFERENCES
  10. Supporting Information

This article describes the synthesis and enzymatic study of newly synthesized analogs of trypsin inhibitors SFTI-1 that were fluorescent labeled on their N-terminal amino groups. Two fluorescent derivatives of benzoxazole (3-[2-(4-diphenylaminophenyl)benzoxazol-5-yl]-l-alanine–[(4NPh2)Ph]Box-Ala and 3-[2-(2',4',5'-trimethoxyphenyl)benzoxazol-5-yl]-l-alanine–[2,4,5-(OMe)3Ph]Box-Ala) were used as efficient fluorescent labels. The compounds obtained preserved their inhibitory activity and were efficient inhibitors of bovine trypsin or chymotrypsin. Nevertheless, their association inhibition constants were one or two orders of magnitude lower than those determined for unlabeled monocyclic SFTI-1 or [Phe5]SFTI-1, respectively. The conjugates obtained were found to be proteolytically stable in the presence of cognate enzymes. Applying such fluorescent peptides, we were able to investigate enzyme-inhibitor complex formation using fluorescent techniques. We found that such compounds were rapidly internalized by the fibroblast or cancer cells with no cytotoxic effects. © 2013 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 102: 124–135, 2014.


INTRODUCTION

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8.  
  9. REFERENCES
  10. Supporting Information

With only one exception, the canonical inhibitors that belong to the Bowman–Birk inhibitor (BBI) family are polypeptides consisting of more than 60 amino acid residues. In 1999, Luckett et al.[1] isolated a trypsin inhibitor SFTI-1 (sunflower trypsin inhibitor 1) from sunflower seeds, and this was the smallest and in addition the most potent of all the inhibitors of the BBI family. SFTI-1 is a 14 amino acid circular peptide that was found to have high homology to the binding loop of other BBI family members. Its primary structure is shown in Figure 1. The reactive site P1-P1′ of the SFTI-1 inhibitor is located between the Lys5-Ser[6] residues and therefore exhibits trypsin-like specificity, possessing a high affinity to trypsin (Ka = 1.1 × 1010 m−1).[2] Owing to its exceptionally small size, compact structure, and high inhibitory activity, SFTI-1 is considered to be a very attractive template for designing proteinase inhibitors against physiologically important enzymes, which could be potentially used as pharmacological agents. Results of extensive studies on SFTI-1 are summarized in review papers.[3]

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Figure 1. Structure of SFTI-1.

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In the mid 1990s, it was shown that short positively charged peptides were able to cross cell membranes.[4] Since then, many examples of such cell penetrating peptides (CPPs) have been reported. Among them are peptides derived from proteins, synthetic chimeras (composed of fragments of endogenous biologically active peptides) and also those with non-naturally accruing (designed) primary structures. CPPs have attracted significant attention due to their ability to deliver cargos, including biologically active agents with different chemical characteristics (proteins, peptides, oligonucleotides, and low-molecular drugs) into the cell. Such bioconjugates have therapeutic or diagnostic agent potentials, which are difficult to overestimate.[5] Another group of peptides extensively studied in last two decades are cyclotides.[6, 7] These plant-derived miniproteins with a cysteine knot arrangement of three conserved disulfide bonds and head-to-tail cyclization are, unlike most peptides, exceptionally resistant to thermal/chemical denaturation, and enzymatic degradation. They possess numerous biological activities, including antimicrobial, antitumor, and anti-HIV. As part of the plant defense system, some of them display toxic effects and insecticidal activity. Recently, it has been shown,[7-9] using confocal fluorescence microscopy imaging, that cycliotides (MCoTI and kalata B1) are able to internalize into the breast cancer cell line, MCF-7, the macrophage cell line, RAW264.7 and HeLa cells. Interestingly, SFTI-1 also internalize into MCF-7 cells, although it does not interact with any of the investigated phospholipids.[10] To monitor the cellular uptake, the peptides were labeled with fluorescence markers, biotin or AlexaFluor488, which were attached to the side-chain amino groups of Lys residues present in the studied peptides. In the case of trypsin inhibitors MCoTI and SFTI-1, Lys residues are located in substrate specific P1 position (3 and 5, respectively). Therefore, such labeled cyclic peptides lost their inhibitory activity. In the studies by Craik's and Camarero's groups, the main goal was to obtain labeled peptides that would be able to cross the cell membranes and they did not focus their attention on the inhibitory activity of these cyclic peptides. We decided to design analogs of SFTI-1, which would combine both properties. Our intention was to obtain fluorescent analogs of SFTI-1 useful for monitoring inhibitor—proteinase complexes formed inside the cells. Such peptides would be a good starting point for the design of fluorescent inhibitors of physiologically important proteinases and they could be utilized to trace these enzymes in biological samples. As a lead structure, we selected monocyclic SFTI-1 with only a disulfide bridge. In our earlier research[2] we had proved that the elimination of head-to-tail cyclization in wild SFTI-1 did not significantly influence the inhibitory activity determined as the equilibrium association constant (Ka) and proteolytic susceptibility. As fluorescent labels, we decided to use (benzoxazol-5-yl)-l-alanine derivatives: 3-[2-(4-diphenylaminophenyl)benzoxazol-5-yl]-l-alanine–[(4NPh2)Ph]Box-Ala[11] and previously unpublished 3-[2-(2′,4′,5′-trimethoxyphenyl)benzoxazol-5-yl]-l-alanine–[2,4,5-(OMe)3Ph]Box-Ala. A series of Box-Ala derivatives have recently been synthesized by the coauthors of this article. Because of their photophysical properties namely high quantum yields and excellent stability,[12] these compounds can be useful as fluorescence probes. The chemical formulas of both fluorophores, together with their maximum wavelengths of excitation and emission are given in Figure 2. To preserve the inhibitory activity of synthesized SFTI-1 analogs, they were coupled through their carboxyl groups to the peptide N-termini. As reported previously such N-terminal modification did not influence potency of inhibitor.[13] The primary structures of synthesized peptides are shown in Figure 3. In two fluorescent analogs (numbered 1 and 2), Phe residue was introduced in substrate specific P1 position. On the basis of our previous reports, this should change the specificity of synthesized peptides from trypsin to chymotrypsin inhibitory activity. To eliminate the possible trypsin cleavage site between Arg[2] and Cys[3], this first amino acid residue was omitted in all synthesized SFTI-1 analogs. To determine the impact on the inhibitory activity of the fluorescent moieties attached to SFTI-1 analogs, two analogs 5 and 6 modified by PEG were also synthesized.

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Figure 2. Benzoxazole derivatives that were used as fluorescent labels with their extinction/emission wavelengths.

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Figure 3. Primary structures of synthesized SFTI-1 analogs.

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MATERIALS AND METHODS

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8.  
  9. REFERENCES
  10. Supporting Information

Chemical Synthesis of Fluorescent Amino Acid Derivates

N-(tert-butoxycarbonyl)-3-[2-(4-diphenylaminophenyl)benzoxazol-5-yl]-l-alanine–Boc-[(4NPh2)Ph]Box-Ala was synthesized from N-Boc-protected 3-amino-l-tyrosine methyl ester, and N-(tert-butoxycarbonyl)-3-[2-(2,4′,5′-trimethoxyphenyl)benzoxazol-5-yl]-l-alanine–Boc-[2,4,5-(OMe)3Ph]Box-Ala from N-Boc-protected 3-amino-l-tyrosine, via the intermediate Schiff base, which underwent oxidative cyclization to the heterocyclic compound in the presence of lead tetra-acetate in DMSO, according to the procedure published previously.[12, 14] This was followed by methyl ester saponification using 1N NaOH in methanol. 3-Nitro-l-tyrosine methyl ester, N-Boc-3-nitro-l-tyrosine methyl ester, and N-Boc-3-nitro-l-tyrosine were prepared according to literature procedures published in Refs. [15] and [16], respectively.

Materials

All protected amino acids were purchased from GLS CHINA, and AEA (5-amino-3-oxapentanoic acid) and ADEA ((2-(2-aminoethoxy)ethoxy) acetic acid) came from Iris Biotech GMBH, Germany. The Bovine α-chymotrypsin and β-trypsin, the chromogenic trypsin burst substrate–nitrophenyl-4′-guanidinobenzonate (NPGB) and ovomucoid from turkey egg whites (OMTKY-3) were purchased from Sigma–Aldrich. The chromogenic turnover substrates: -benzoyl-d,l-arginine 4-nitroanilide (BAPNA), Suc-Ala-Ala-Pro-Phe-pNA, and Suc-Ala-Ala-Pro-Leu-pNA were purchased from Bachem, Switzerland, while Z-Phe-Ala-Thr-Tyr-ANB-NH2 was synthesized according to Wysocka et al.[17] All UV–VIS measurements were performed using a Cary 3E Spectrophotometer (Varian, Australia).

Peptide Synthesis

All the peptides were synthesized manually following the solid-phase method using Fmoc chemistry. The following amino acid derivatives were used: Fmoc-Gly, Fmoc-Cys(Trt), Fmoc-Thr(tBu), Fmoc-Ser(tBu), Fmoc-Lys(Boc), Fmoc-Ile, Fmoc-Pro, and Fmoc-Phe, Fmoc-Asp(OtBu). The protected derivative of C-terminal amino acid residue, Fmoc-Asp(OtBu), was attached to the 2-chlorotritylchloride resin (substitution of Cl 1.46 mequiv/g) (Calbiochem-Novabiochem AG, Switzerland) in the presence of an equimolar amount of DIPEA (based on the amino acid) under anhydrous conditions in DCM solution. Peptide chains were elongated in consecutive cycles of deprotection and coupling. Deprotection was performed with 20% piperidine in DMF/NMP (1:1, v/v) with the addition of 1% Triton X-100, whereas chain elongation was achieved with standard DIC/HOBt chemistry; 3 equiv of protected amino acid derivatives were used. The same method was applied for coupling Fmoc-PEG derivatives and fluorescent compounds (used as N-protected Boc derivatives). After completing the syntheses, the peptides were cleaved from the resin simultaneously with the side chain deprotection groups in a one-step procedure, using a TFA/phenol/triisopropylsilane/H2O (88:5:2:5, v/v/v/v) mixture. In the last step, the disulfide bridge was performed using a 0.1M methanolic iodine solution, following the procedure described elsewhere.[18] The crude peptides were purified using HPLC on a Beckman Gold System (Beckman) with an RP Kromasil-100, C8, 5 μm column (8 × 250 mm) (Knauer, Germany). The solvent systems were 0.1% TFA (A) and 80% acetonitrile in A (B). Either isocratic conditions or a linear gradient were applied (flow rate 3.0 ml/min, monitored at 226 nm). The purity of the synthesized peptides was checked on an RP Kromasil 100, C8, 5 μm column (4.6 × 250 mm) (Knauer, Germany). The solvent system was as above. Linear gradient from 10 to 90% B for 30 min, flow rate 1 ml/min, monitored at 226 nm. The mass spectrometry analysis was carried out on a MALDI MS (a Biflex III MALDI-TOF spectrometer, Bruker Daltonics, Germany) using a-CCA matrix.

Determination of Association Equilibrium Constants

The bovine β-trypsin (Sigma Chem) concentration was determined using spectrophotometric titration with 4-nitrophenyl-4′-guanidinobenzoate (NPGB) at an enzyme concentration oscillating around 10−6 M. A standardized trypsin solution was used to titrate the ovomucoid from turkey egg whites, which in turn served to determine the solution concentration of the bovine α-chymotrypsin (Sigma Chem). The concentrations of the SFTI-1 analogs were determined via titration of their stock solutions with standardized bovine β-trypsin and bovine α-chymotrypsin with BAPNA and Suc-Ala-Ala-Pro-Leu-4-nitroanilide, respectively. The association constants were measured using the method developed in the laboratory of Empie and Laskowski.[19, 20] Enzyme–inhibitor interactions were determined in a 0.1M Tris–HCl (pH 8.3) buffer containing 20 mM CaCl2 and 0.005% Triton X-100 at room temperature. Increasing amounts of the inhibitor, varying from 0 to 2E0 (E0 is the total enzyme concentration), were added to a fixed amount of the enzyme. After a 3 h of incubation, residual enzyme activity was measured using the turnover substrate. The measurements were carried out at initial enzyme concentrations over the ranges 1.3–9.1 nM for trypsin and chymotrypsin, respectively. The residual enzyme activity was measured with BAPNA and Z-Phe-Ala-Thr-Tyr-ANB-NH2 [17] as chromogenic substrates for trypsin and chymotrypsin, respectively. In all cases, the initial substrate concentration was below 0.1 KM. The experimental points were analyzed by plotting the residual enzyme concentration [E] versus the total inhibitor concentration [I0]. The experimental data were fitted to the theoretical values using the GRAFIT software package.[21] A more detailed procedure was described in our recent article.[22] The inhibitory activities, expressed as the Ka values with bovine β-trypsin or bovine α-chymotrypsin for the SFTI-1 analogs, are summarized in Table 1.

Table 1. Physicochemical and Inhibitory Activity of the Compounds Studied
Analog MW Calc./(found)RT (min)Ka (M−1)
  1. a

    Association constant (Ka) values determined for (T) with bovine β-trypsin, (CH) with bovine α-chymotrypsin.

SFTI-1 wild 1513.8 (1513.4)16.71(1.1 ± 0.2) × 1010 (T)1
SFTI-1 (monocyclic) 1531.8 (1531.2)18.15(9.9 ± 1.1) × 109 (T)
[Phe5]SFTI-1 1550.2 (1550.8)20.64(2.0 ± 0.2) × 109 (CH)
[desArg2]SFTI-1 1375.6 (1375.4)17.56(4.6 ± 0.3) × 108 (T)
[desArg2,Phe5]SFTI-1 1394.0 (1394.3)19.11(1.0 ± 0.1) × 109 (CH)
[(4-NPh2)Ph]Box-Ala-ADEA-[desArg2,Phe5]SFTI-1(1)1972.1 (1971.5)30.70(5.0 ± 0.4) × 107 (CH)
[2,4,5-(OMe)3Ph]Box-Ala-AEA-ADEA-[desArg2,Phe5]SFTI-1(2)1995.8 (1995.6)23.62(4.9 ± 0.3) × 107 (CH)
[(4-NPh2)Ph]Box-Ala-ADEA-[desArg2]SFTI-1(3)1953.1 (1952.8)29.25(1.5 ± 0.2) × 109 (T)
[2,4,5-(OMe)3Ph]Box-Ala-AEA-ADEA-[desArg2]SFTI-1(4)1976.7 (1976.8)19.51(1.5 ± 0.1) × 109 (T)
ADEA-[desArg2,Phe5]SFTI-1(5)1540.7 (1540.8)16.12(1.1 ± 0.1) × 109 (CH)
ADEA-[desArg2]SFTI-1(6)1521.7 (1521.9)14.34(1.24 ± 0.2) × 109 (T)

Cell Permeability Assay

The 1.5 × 106 of cells (MOLT-4: human acute lymphoblastic leukemia cell line), immortalized human fibroblast cell line (46BR.1N) and MCF-7 cell line were cultured at 37°C in a humidified atmosphere saturated with 5% CO2 in DMEM medium. Next cell were resuspended or cultured in 3 ml of DMEM (Modified Eagle's Medium) and 5 µl of inhibitors 1–4 at two different concentration 10 µM and 25 was added and mixed thoroughly. At the indicated time points (5, 30, and 60 min), the 0.5 × 105 cells from each system were collected and washed 3 times with DMEM medium supplemented with TRITON 0.2% and finally resuspended in 100 mM PBS with 2% FBS and subjected for further studies.

Fluorescence Microscopy

Harvested by trypsinization and counted cells were seeded on Millicell EZ Slide (8-well, EMD Milipore, Warsaw, Poland) at a density of 5 × 104 cells/well and left to attach overnight. Next, the cells were treated with analogs at different concentrations (25 and 10 µM), after aforementioned time points were washed twice using 100 mM PBS, fixed using Cytofix (Sanko, Klembow, Poland) and immediately analyzed using Olympus IX81 equipped with Lumen Dynamics X-Cite Series 120 PC Q lamp module.

Flow Cytometry

Briefly, cell were harvested by trypsinization and counted. Next cell were seeded on 24-well plates (Corning, Costar, Amsterdam, Netherlands) at a density of 105 cells/well and left to attach overnight. Subsequent, the cells were treated with tested compounds at different concentrations (25 and 10 µM) or benzoxazole alanine derivates (0.273 µM), washed twice with 100 mM DMEM medium with 2% FBS (HyClone, ThermoScientific, Brookfield, WI), harvested by trypsinization, and suspended in 100 mM PBS with 2% FBS. The flow cytometry analysis was performed immediately using BD LSRFortessa cytometer (BectonDickinson, San Jose, CA) exploiting UV laser (λext = 355 nm, λem = 450/50 nm). Data analysis was conducted using FACSDiva software (BectonDickinson, San Jose, CA). Presented data represents two independent experiments performed in duplicate and shows the relative fluorescence increase ± SD. Histograms present the results of one representative experiment.

Complex Enzyme-Inhibitor Investigations

The inhibitors (2 and 4) stock solutions at final concentration 1 × 10−4 M were used. Enzymes (trypsin or chymotrypsin) stock solution were 3 × 10−4 M were used. For enzyme-inhibitor studies 20 µl of inhibitor (5 × 10−5 M) and 10 µl (2 × 10−5 M) of enzyme was mixed in the 0.1M Tris-HCl buffer, pH 7.5, and incubated 20 min. Nonlabeled monocyclic SFTI-1 concentration was 1.6 × 10−3 M and 50 µl (2 × 10−4 M) of such solution was added into above mixtures. The studies were performed by means of gel filtration chromatography on an HPLC (Jasco) with an FP2020 (Jasco, Japan) scanning fluorescence detector equipped with an (10 × 260 mm) Agilent Zorbax Bio Series, GF 250 column. As the eluent, a 0.1M solution of Tris-HCl buffer, pH 7.5 with flow rate 1 ml/min was used. The extinction/emission wavelengths were as follows: inhibitor 1 and 3 320 nm/400 nm inhibitor 2 and 4 380 nm/460 nm.

RESULTS

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8.  
  9. REFERENCES
  10. Supporting Information

Both fluorescent markers [(4NPh2)Ph]Box-Ala and [2,4,5-(OMe)3Ph]Box-Ala were attached as N-protected Boc derivatives to the N-termini of synthesized peptides using the standard method (DIC/HOBt) utilized in solid-phase peptide synthesis. In our previous study,[23] we reported that, in monocyclic SFTI-1 analogs, the peptide bond Arg2-Cys[3] was cleaved upon incubation with trypsin; therefore, in order to prevent splitting of the fluorescent moiety in all the synthesized peptides, the Arg2 was removed. Such modification together with the introduction of a PEG linker (ADEA) at the N-termini of the synthesized peptides did not influence the inhibitory activity. Both reference compounds 5 and 6 appeared to be potent trypsin and chymotrypsin inhibitors, respectively. The determined Ka values were the same order of magnitude as patent monocyclic SFTI-1 and [Phe5]SFTI-1, respectively (see Table 1). All synthesized fluorescence labeled compounds was also efficient inhibitors of bovine trypsin (3, 4) and chymotrypsin (1, 2). Introduction of a fluorescent benzoxazole moiety into trypsin inhibitors did not reduce their potency, since their association constants were in the same range as monocyclic SFTI-1 (109 M−1). In the case of compounds 1 and 2 (designed as chymotrypsin inhibitors), a significant reduction of inhibition potency (100 times) was observed, in relation to the parent compounds, [Phe5]SFTI-1 and 5.

All fluorescent peptides incubated with appropriate enzymes (trypsin or chymotrypsin) remained proteolytically stable for 24 h (see Figure 4). Also, MS analysis indicated that no degradation is observed over this period (data not shown).

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Figure 4. HPLC analysis of compounds 1–4 incubated with bovine β-trypsin in Tris-HCl pH 8.3. Two time points were used solid line-time 0 and dash line 24 h. UV detection was applied at 226 nm.

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Bearing in mind the excellent proteolytic stability and efficient inhibition of cognate enzymes displayed by the obtained labeled inhibitors, we decided to monitor the protease-inhibitor complex using the chromatography technique. In this experiment, in order to form a complex, trypsin (at concentration of 2.09 × 10−5 M) and inhibitor 4 (5.06 × 10−5 M) were mixed in the appropriate buffer (Tris-HCl, pH 8.3). An analysis was performed using an HPLC system equipped with a size exclusion column and the fluorescent intensity was followed. As presented in Figure 5A, inhibitor 4 itself has a retention time 9.24 min. After 15 min of incubation with bovine β-trypsin, a new peak with a retention time of 6.94 min, significantly different from the elution time of the inhibitor alone, appeared and this was recognized as a noncovalent enzyme-inhibitor complex. This peak was not visible in the chromatogram when trypsin was first incubated with peptide 4 and after 15 min nonlabeled monocyclic SFTI-1 (1.6 × 10−4 M) was added to this mixture. A similar effect was observed in the case of a system where a trypsin-inhibitor 4 complex had previously formed and such a mixture was acidified to obtain pH 3. In this case, the peak with a retention time of 7.05 min (corresponding to an enzyme-inhibitor complex) was significantly reduced and one denoted a free inhibitor (retention time 9.29 min) appeared. This is in close agreement with well-known data that for canonical inhibitors: the strength of their complexes with cognate enzymes decreases with decreases in the pH.

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Figure 5. Size exclusion chromatography: (A) analog 4 (B) analog 4 incubated with bovine β-trypsin in tris-HCl buffer pH 8.3, (C) analog 4 incubated with bovine β-trypsin in tris-HCl buffer pH 7.5, and then acidified to pH 3. (D) Analog 4 incubated with bovine β-trypsin in tris-HCl buffer pH 8.3 followed by with a 5M excess of monocyclic SFTI-1. The fluorescence detector was used with excitation wavelength at 320 nm and emission wavelength at 400 nm.

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Less apparent observation was made for a mixture of inhibitor 2 (concentration 5.01 × 10−5 M) and bovine α-chymotrypsin (2.1 × 10−5 M). The retention time of inhibitor 2 is 7.53 min. As a result of the incubation of these two constituents in Tris HCl buffer at pH 7.5, multiple peaks were observed between 6.18 and 10.34 min. Among them the most intense had a retention time of 6.59 min (Figure 6). Such results could be an effect of the heterogeneity of the enzyme solution because of self-degradation of chymotrypsin in solution. In the presence of a 5 M excess of [Phe5]SFTI-1 (concentration 1.02 × 10−4 M) all of the aforementioned peaks disappeared and only one, with a retention time of 7.53, corresponding to inhibitor 2 was visible.

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Figure 6. Size exclusion chromatography: (A) analog 3, (B) analog 3 incubated with bovine β-trypsin in tris-HCl buffer pH 8.3, (C) analog 3 incubated with bovine β-trypsin in tris-HCl buffer pH 8.3 with a 5M excess of monocyclic [Phe5]SFTI-1. The fluorescence detector was used with excitation wavelength at 320 nm and emission wavelength at 400 nm.

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Peptides 1 and 3 at the concentration of 2.53 × 10−5 M and 2.56 × 10−5 M, respectively, were analyzed using the condition described above. Again, only fluorescent molecules were detected. When compared to peptides 2 and 4, a surprisingly short retention time, equal to 4.52 min, was recorded for both compounds. The difference between compound 1 and 2 is just a fluorophore labeling (a 200 Da molecular weight increase); thus, such a shift in retention times was not justified. The addition of either appropriate proteinases or pH change did not influence the retention time of the analyzed inhibitors. Repeating the analysis in 6M urea present in both sample and elution buffer changed the retention time for both compounds (1 and 3) dramatically from 5 to 9 min (Figure 7). This indicated that both compounds in separation conditions form aggregates and so were not utilized in further size exclusion experiments. It worth underlining that such an observation was not for compounds 2 and 4 in the presence of a denaturing agent.

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Figure 7. Size exclusion chromatography: (A) analog 1 in tris-HCl buffer pH 7.5. (B) Analog 1 incubated and analyzed in presence of 6M urea. The fluorescence detector was used with excitation wavelength at 380 nm and emission wavelength at 460 nm.

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Incubation of compounds 1–4 with cancer cell lines (MOLT-4 and MCF-7) or transformed fibroblast cell lines at micromolar concentrations, displayed a noncytotoxic effect over 24 h (data not shown). Incubation of all the obtained compounds results in their visible presence within the cells used. Each cell system was incubated with an appropriately labeled peptide in DMEM medium and extensively washed by PBS supplemented with Triton X-100. Similar results were obtained for all tested compounds.

On Figure 8 as the results of incubation of inhibitors 1–4 (10 × 10−6 M) with MCF-7 cell line is presented. There is a clear presence of fluorescent compounds inside the cell that is localized outside the nucleus. Similar observations were recorded for other cell line tested (MOLT-4 cancer cells and regular human fibroblast) see Figure 1 Supporting Information.

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Figure 8. Transmitted light and fluorescent images and its overlays of MCF-7 cells incubated with compounds 1–4: Incubation time 60 min and magnification ×400. (A) Analog 1, (B) Analog 2, (C) Analog 3, and (D) Analog 4. All compounds at concentration 10 µM.

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Incubation of aforementioned cell line with fluorophore moieties namely [(4NPh2)Ph]Box-Ala and [2,4,5-(OMe)3Ph]Box-Ala at concentration 0.256 µM also results in their fast internalization within the cell cytoplasm. We observed no cytotoxic effect during duration of the experiment. We have reason to believe that excellent permeability of labeled inhibitors is due to presence of benzoxazole alanine derivates present on their N-termini.

Further flow cytometry analysis of cells used in a previous experiment indicates that the majority of the cells are stained and highly fluorescence as compared to cells that were not incubated with the labeled SFTI-1 analogs. Moreover, their fluorescence does not depend on the time of incubation or on temperature. Fluorescence intensity and the amount of stained cells remain at the same high level as compared to the negative nonstained control (Figure 9).

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Figure 9. Flow cytometry analysis of MCF-7 cell line: (A) analog 1, (B) analog 2, (C) analog 3, (D) analog 4. The excitation wavelength was λext = 355 nm and emission, λem = 450/50 nm. Two concentrations of tested compounds were used (25 and 10 µM). Incubation time 60 min.

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DISCUSSION

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8.  
  9. REFERENCES
  10. Supporting Information

Inhibitory Properties

Fluorescent Boc-[(4NPh2)Ph]Box-Ala and [2,4,5-(OMe)3Ph]Box-Ala. were synthesized from N-Boc-protected 3-amino-l-tyrosine methyl ester using previously described procedures. The obtained peptide conjugates appeared to be strong inhibitors of cognate enzymes despite the attached fluorophores and the length of PEG derivates (see Table 1). Association constants of compounds 3 and 4 with bovine β-trypsin are in the same range (109 M−1). Such values are one range less than native SFTI-1, but similar to the inhibitor potency of the monocyclic inhibitor and even higher than that obtained for pegylated [desArg2]SFTI-1 analog (compound 6). A similar situation is observed for their chymotrypsin counterparts, peptides 1 and 2, but a lower associated constant (107 M−1) as compared to [Phe5]SFTI-1 and its pegylated analog (compound 5) was obtained. All compounds (1–4) incubated with an appropriate enzyme (trypsin or chymotrypsin) remain proteolytically stable for 24 h (Figure 4) and MS analysis indicates that no degradation is observed during this time. This is an interesting finding since, for the monocyclic SFTI-1 molecule, the peptide bond Lys5-Ser5 is slowly hydrolyzed until the equilibrium is reached between open and intact forms (1:9). We believe that such behavior is observed because of the presence of a bulky hydrophobic group at the N-terminal part of the inhibitor.

Cell Studies

All four compounds, when incubated with MOLT-4 and fibroblast lines at micromolar concentration, displayed no cytotoxic effect over 24 h. It worth emphasizing that bezoxazole moieties display moderate to high cytotoxicity against various cell lines (unpublished data Guzow) at millimolar concentration. However, when conjugated with SFTI-1 such an effect is significantly reduced. Those compounds were found to be cell permeable at submicromolar concentration (Figure1 in Supporting Information).

All peptide conjugates (1–4) are efficiently internalized into the living cancer (HELA and MCF-7) or healthy cells (human fibroblasts) and judging on the fluorescent microscope picture they are mainly localized in the cytoplasmic fraction (Figure 6 and Figure 1 Supporting Information). Further flow cytometry analysis indicates (Figure 9 and Figure 2 Supporting Information) that such cell permeability is dose dependent when concentrations from 3.14 µM to 31.4 nM of compounds are used. Presence of macropinocytotsis inhibitor (amiloride at 1.25 mM) did not influence such uptake. Temperature did not affect membrane crossing efficiency, which remains at similar levels for systems incubated at 4 and 37°C (Figure 10). All of above results suggest that this process is passive and not governed by the cell. These results do not correlate well with data provided by Cascales et al.[10] for the aforementioned selected cyclic peptides, also referred to as cyclotides.[18] In their study, they incubated three groups of fluorescent labeled cyclic peptides, the sequences of which were derived from leading structures: MCoTI-II, kalata B1, and SFTI-1. To all peptides, either biotin moiety or ε-amino groups of Lys were conjugated with Alexa 488 fluorophore. Incubation of the obtained fluorescent peptides with phospholipids and further with a transformed macrophage cell line (RAW) and a breast cancer cell line (MCF-7) indicates that MCoTI-II and kalata-B1 derived fluorescent peptides were internalized into the cell due to strong interaction with the phospholipids, as a model of the cell membrane. The authors postulated that such massive uptake of the studied fluorescent peptides is due to vesicle formation of the membrane bound peptides and its engulfment into the cell, called the macropinocytosis phenomenon. Such results were not observed in the case of Alexa-SFTI-1 conjugates that display no affinity for membrane mimicking phospholipids. A limited amount of labeled SFTI-1 was identified inside the cell but the mechanism of its presence is unknown.

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Figure 10. Time dependence cell permeability (MCF-7) assay for compounds 1–4. The time points for time-dependent cell permeability assay were 5, 30, and 60 min. Additionally, two different temperatures 36 and 4°C were applied and incubated for 30 min. Peptide concentration 10 µM. Amiloride was used at concentration of 1.25 mM.

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Figure 11. Dose and time dependence cell permeability (MCF-7) assay for compounds 1–4. The time points for time-dependent cell permeability assay were 5, 30, and 60 min. Additionally, two different temperatures 36 and 4°C were applied and incubated for 30 min. Concentration of the compounds is 0.273 µM. Amiloride was used at concentration of 1.25 mM.

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Such a discrepancy between SFTI-1 ability to cross the cell membrane could be explained by the nature of the fluorophore used. Benzoxazole derivatives that were attached to the N-terminal of SFTI-1 and its analog [Phe5]SFTI-1 are much more hydrophobic than those fluorophores used in the Australian group's studies. Both benzoxazole derivates penetrate the cell in submicromolar concentration that is much lower than corresponding SFTI-1 conjugates (Figure 11). This indicates that nature of these large hydrophobic groups is the main factor, which enhances cell permeability of obtained compounds (1–4).

Size Exclusion Chromatography

Having obtained fluorescent inhibitors of proteolytic enzymes, we decided to investigate if we could utilize their fluorescent properties and monitor the inhibitor-enzyme complex formation. We wanted to test if the obtained compounds could act as potential noncovalent activity-based probes. Since the inhibitor interacts only with native active proteins, this should be especially interesting in the context of following protease activity. Moreover, SFTI-1 molecules display inhibitory activity/properties against a cohort of trypsin-like proteases, including trypsin, matriptase, tryptase, and many more.[3] The reporter molecules (benzoxazoles) are able to produce a strong fluorescent signal detectable by simple UV irradiation. Additionally, it is widely known that, in the case of reversible proteinase-inhibitor complexes, post detection pH change yields strip off the inhibitor(s) yielding the recovery of pure proteinase. To investigate their ability to form and visualize enzyme inhibitor complexes, we decided to utilize gel filtration chromatography with fluorescence detection mode. As mentioned earlier, we were able to obtain a clear picture only for two SFTI-1 derivatives. Two other peptides (1 and 3) formed aggregates under assay conditions and were not subjected to further studies. Incubation of peptides 2 or 4, both labeled with chymotrypsin or trypsin, respectively, result in the formation of a new signal corresponding to a noncovalent enzyme-inhibitor complex. Such a signal was reduced or diminished when nonlabeled SFTI-1 or its analog with chymotrypsin specific [Phe5]SFTI-1 was present in the solution. The same affect was observed in the case of alerting the pH of such mixture due to a disruption of the complex. Utilization of SFTI and its analogs was described by Pereira et al., showed that immobilized monocyclic SFTI-1 analogs modified in the P1 position on agarose gel.[24] The covalent linkage was achieved by the reaction of an aldehyde moiety of an oxidized Ser residue, additionally attached at the peptide N-terminus, and a hydrazine group of agarose. It was demonstrated that these immobilized SFTI-1 analogs could serve as affinity probes for the isolation of serine proteinases with different specificities.

CONCLUSION

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8.  
  9. REFERENCES
  10. Supporting Information

Four noncytotoxic cell permeable SFTI-1 analogs conjugated with fluorogenic moieties of benzoxazoles were obtained. Introduction of N-terminal fluorogenic groups did not influence their interaction with cognate proteinases, but resulted in an increase in their proteolytic stability and cell permeability, when compared to the parent compounds. We believe that such conjugates could serve as reversible activity-based probes and could be utilized in the study of protease inhibitor interactions.

 

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8.  
  9. REFERENCES
  10. Supporting Information

Authors would like to thank the Polish National Science Centre. Grant Number: UMO-2011/01/B/ST5/03772 (KR) and NeoLek Laboratory cofinanced by The European Regional Development Fund POIG.02.01.00–02-073/09 for performing of culture cell analyses (MP).

REFERENCES

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8.  
  9. REFERENCES
  10. Supporting Information

Supporting Information

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8.  
  9. REFERENCES
  10. Supporting Information

Additional Supporting Information may be found in the online version of this article.

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