Functional Analysis of Novicidin Peptide: Coordinated Delivery

Article Cite This: Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Functional Analysis of Novicidin Peptide: Coordinated Delivery System for Zinc via Schiff Base Ligand Vedran Milosavljevic,†,‡ Yazan Haddad,†,‡ Amitava Moulick,†,‡ Hana Buchtelova,†,‡ Roman Guran,†,‡ Tomas Pospisil,§ Kamila Stokowa-Sołtys,∥ Zbynek Heger,†,‡ Lukas Richtera,†,‡ Pavel Kopel,†,‡ and Vojtech Adam*,†,‡ †

Central European Institute of Technology, Brno University of Technology, Purkynova 123, 612 00 Brno, Czech Republic Department of Chemistry and Biochemistry, Mendel University in Brno, Zemedelska 1, 613 00 Brno, Czech Republic § Department of Chemical Biology and Genetics, Centre of the Region Hana for Biotechnological and Agricultural Research, Faculty of Science, Palacky University, Slechtitelu 241/27, 783 71, Olomouc, Czech Republic ∥ Faculty of Chemistry, University of Wrocław, Joliot-Curie 14, 50-383 Wrocław, Poland

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‡

S Supporting Information *

ABSTRACT: Novicidin (NVC), is a membrane-penetrating peptide, which forms a stable complex with Zn-Schiff base with interesting antitumor selectivity. We studied NVC derivatives to determine functional roles of key amino acids in toxicity, helicity, and binding of the Zn-Schiff base complex. Trimmed derivatives highlighted the role of peptide length and helicity in toxicity and membrane penetration. The removal of Lys from position 1 and 2 strongly increases the ability to disrupt the membranes. The trimming of the N-terminal residues significantly increases the stability of peptide helicity enhancing penetrating properties. Gly residue derivatives undermined a role of peptide bending in membrane penetration and toxicity. After the substitution of the central Gly derivatives with Ile or Lys, the peptides retained toxicity. These results illustrate the minor role of central helix bending in NVC toxicity. Binding-site-peptide derivatives identified His residue as the sole Zn-Schiff base binding site and eliminated the role of other aromatic residues.

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INTRODUCTION Cell penetrating peptides (CPPs) are usually isolated from protein transduction domains or membrane translocation sequences.1 Their origin enables them to interact/bind with membrane surface protein, for instance, glycosaminoglycan.2,3 Due to that, CPPs are capable of participating in a diverse range of cellular functions, such as signaling pathways, drug carriers, depolarization, or leakage of cell membrane or cell death.4−6 Biological activity of CPPs is closely connected with their physicochemical properties.7 For instance, the lytic nature of antimicrobial peptides (AMPs) comes from amphipathic secondary structure, where residues are segregated into hydrophobic and cationic domains. This plays a major role in membrane anchoring and disruption. AMPs will preserve their lytic nature only if the amphipathic structure remains unchanged.8−11 This indicates that diverse functions of CPPs are strongly dependent on their structure. On the other hand, entirely new peptide functions can be obtained only by modification of existing structure which is the essential strategy for peptide evolution.12,13 Diversity of peptides can be severely affected by deletion of residues or by single point mutation of known sequences.14,15 However, amphipathic structure is not always required for optimal activity and specificity. 16 Intervention to the cationic or hydrophobic domain through © XXXX American Chemical Society

substitution of amino acids often leads to disruption of the amphipathicity of a helix. It has been suggested that disruption of the amphipathicity usually contributes to the strong antimicrobial activity by preservation of peptide helicity in the case of AMPs.17−19 An alternative route for maintenance of peptide helicity can be found in the interaction of peptide with free metal ions. The interaction of metal with side chain usually constrains the geometry of the peptide. In the presence of free metal ions, the side chain of the peptide starts to behave as a ligand, resulting in free metal-ion inducing α-helix formation.20,21 The ideal choice can be found in zinc(II) ions due to their essential role in various cellular activities such as cell growth, enzyme activity, and reduction of mitochondrial aconitase activity.22 However, modifications of the peptide with zinc(II) ions usually have limitations with respect to competition among biomacromolecules for zinc(II) ions.23 Using a Schiff base as a carrier for metal ion can limit the competition of other molecules from zinc(II) ions. The coordination of transition metal ions to Schiff bases and their interaction with peptide leads to stabilization of the entire Received: May 28, 2018 Revised: August 2, 2018 Published: August 7, 2018 A

DOI: 10.1021/acs.bioconjchem.8b00370 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Bioconjugate Chemistry

membrane destructive nature of NVC peptide can be achieved by selective replacements or permanent deletion of the amino acid residues. Sequence modification can lead to design of nontoxic peptide with retained penetrating capabilities.15 In our previous study, we found that the NVC peptide has strong anticancer activity but also a severe cytotoxic effect on normal healthy cells. However, treatment of healthy cells with an NVC-Zn Schiff base complex revealed a significant decrease in cytotoxicity, while in the case of cancer cells the cytotoxicity was preserved like in the case of NVC peptide. While investigating the effect of oxidative stress to cell lines we discovered that Zn-Schiff base is not the main toxic component of the complex.31 Consequently, our further investigation was focused on determination of the functional roles of NVC peptide substructural elements in the NVC-Zn Schiff base complex. Here, we provide functional dissection of NVC through study of mutant derivatives. We have studied the role of helicity, exemplified by trimming and central Gly peptide derivatives, in controlling membrane penetration and cytotoxic abilities. To study the binding site of Zn-Schiff base, aromatic residues were substituted in binding site peptide derivatives. The current investigation effort revealed that Zn-Schiff base binds specifically to His residue, and thus provides solid ground for development of more potent and selective therapeutics in the future.

Table 1. Molecular Masses of NVC Peptides and NVC Peptides Conjugated with Zn Shiff Base Complex MALDI TOF MS peptide MW (Da)

peptide modified with Zn-S-5 MW (Da)

NVC−2295.491 NVC1−2167.325 NVC2−2053.231 NVC3−1940.231 NVC4−1784.053 NVC5−1627.976 NVC6−1514.950 NVC7−1400.759 NVC8−1226.693

NVC−Zn-S-5−2458.566 NVC1−Zn-S-5−2330.407 NVC2−Zn-S-5−2216.297 NVC3−Zn-S-5−2130.254 NVC4−Zn-S-5−1974.124 NVC5−Zn-S-5−1791.038 NVC6−Zn-S-5−1678.030 NVC7−Zn-S-5−1563.807 NVC8−Zn-S-5−1389.765

complex.24 On the other hand, metal ion coordinated to Schiff base can enhance cytotoxicity and selectivity to the cancer cell lines.25,26 Novicidin (NVC) is a promising candidate for structural investigations due to several properties, such as its cellpenetrating abilities, a low hemolytic effect, a highly amphipathic α-helix structure, and high affinity for anionic lipids that are characteristic of cancer cells. NVC was developed by double mutation of ovispirin-1 (peptide derived from the sheep cathelicidin SMAP-29), at sequence position I10G and G18F. The design of this peptide was focused on highly antimicrobial activity and reduced cytotoxicity.27 It has been reported that NVC is randomly coiled in solution with low helicity. However, in the presence of anionic lipids or organic alcohols such as trifluoroethanol (mimicking the lipid− water interface), it increases the level of helicity. This subsequently causes membrane perforation and formation of transient pore, which leads to leaking of cell contents and finally to a death.28−30 Although NVC has many positive features, treatment of normal human cells with NVC results in significant cytotoxicity.31 A possible strategy for reducing the

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RESULTS

The development of a zinc delivery system for treatment of prostate cancer requires a shuttle that can bring the Zn(II) ions to the cancer cells. The NVC-Zn-S-5 complex is a coordinated, i.e., noncovalently bonded, delivery system.31 Using variants of the NVC peptide can highlight the functional roles related to peptide toxicity and mechanisms of coordination between the peptide, Schiff base, and zinc cargo. Three types of peptide derivatives were investigated here: (1) trimming derivatives, (2) central glycine derivatives, and (3) binding-site derivatives.

Table 2. Analysis of Helix Structure by Different Methodsa MDb

CDc

CDd

FT-IRe

peptide

helices (α, 3−10, pi)

other

α helix

β sheet

α helix

β sheet

helix in amide I

helix in amide II

NVC NVC-1 NVC-2 NVC-3 NVC-4 NVC-5 NVC-6 NVC-7 NVC-8 NVC-Zn NVC-1-Zn NVC-2-Zn NVC-3-Zn NVC-4-Zn NVC-5-Zn NVC-6-Zn NVC-7-Zn NVC-8-Zn

43.3 25.9 25.4 26.7 23.5 29.4 30.6 29.5 31.8

26.4 25.2 23.2 25.6 28.4 23.3 24.8 28.2 17.5

N/A 38.4 56.3 56.3 38.4 38.4 17.9 18.1 18.1 36.4 17.9 36.4 36.4 7.9 17.9 17.9 1.8 9.5

N/A 9.25 5.7 5.7 9.3 9.3 26.4 26.6 26.6 9.68 26.4 9.7 9.7 29.2 26.4 26.4 47.7 33.0

N/A 26.9 26.1 29.5 26.4 26.5 9.1 14.2 8.8 17.3 12.5 17.3 16.9 4.7 8.9 3.9 3.2 3.2

N/A 11.6 11.3 8.7 10.4 11.2 15.5 10.5 13.1 14.3 15.7 14.3 15.3 9.3 15.3 18.4 17.8 15.6

37 100 25 0 0 0 0 0 0 10 4 12 0 0 0 0 0 0

90 49 88 83 1 3 0 96 0 90 92 41 68 13 2 13 96 0

a All values are presented as percentages; interpretation for each method is described in text. N/A: not applicable due to insufficient database spectra. bPercent average secondary structure per residue. cSecondary structure analysis by K2D2.34 dSecondary structure analysis by K2D3.35 e Estimated Gaussian area percentage.

B


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Figure 1. CD spectra of NVC and its derivatives before and after interaction with Zn-S-5 complex. CD spectroscopy showed a decrease in α-helix structure with each trimming. The addition of Zn-S-5 clearly decreased the α-helix structure as demonstrated by red-shifting of spectra. (A) NVC and NVC−Zn-S-5 complex, (B) NVC1 and NVC1−Zn-S-5 complex, (C) NVC2 and NVC2−Zn-S-5 complex, (D) NVC3 and NVC3−Zn-S-5 complex, (E) NVC4 and NVC4−Zn-S-5 complex, (F) NVC5 and NVC5−Zn-S-5 complex, (G) NVC6 and NVC6−Zn-S-5 complex, (H) NVC7 and NVC7−Zn-S-5 complex, and (I) NVC8 and NVC8−Zn-S-5 complex. The indications of α-helix are shown on the graph by positive Cotton effect peak (†) near 192 nm and negative Cotton effect reverse peaks (‡) at 209 and 222 nm.

to belong to the fragments of the complex. Kulac et al.32 reported that high intensity of laser results in departure of O2 molecules from different metal salts including Zn(II) ions, resulting in separation of ions and ligands. Furia et al.33 confirmed that intensity of laser fragmented the metal ion peptide complexes. They found that metal peptide complexes tend to be reduced under laser exposure resulting in ion fragmentation due to the partial decomposition of the complexes. Our results suggested that the present fragments may be due to decomposition of the complexes under the high intensity of the laser and the robustness of the crystals that form the matrix. Further structural study of NVC peptides was conducted by circular dichroism (CD) and Fourier-transform infrared (FT-IR) spectroscopy. CD spectroscopy showed a decrease in α-helix structure with each trim (Figure 1). The addition of Zn-S-5 clearly decreased α-helix structure as demonstrated by a red-shift of spectra. A smooth spectrum was difficult to obtain for the three shortest trimming derivatives (Figure 1G−I). Knowledge-based methods were used to estimate α-helix and β-sheet content from CD spectra (Table 2). Due to the lack of reference structures, the estimation was error prone for the original NVC peptide. However, the decrease in α-helix structures in the rest of trimming derivatives correlated with MD findings. FT-IR showed indications of amide I band α-helix fingerprints near 1655 cm−1 in NVC, NVC-1, and NVC-2 only (Figure 2A−C and Table 2). The addition of Zn-S-5 Schiff base decreased αhelix structure in these three peptides. The rest of the vibrational structures represented in the amide I band were

Trimming Peptide Derivatives. Single molecule molecular dynamics (MD) simulations of NVC and trimmed NVC peptides show a hypothetical NVC structure of two helices separated by a flexible Gly bend and terminated by a double Lys turn and flexible Tyr and Phe in the C-ter (Figures S1A and S2A). The trimming of the N-terminal residues significantly destabilized the N-terminal helix without affecting the C-terminal helix structure (Figures S1 and S2). Root mean square deviation (RMSD) from the equilibrated structure of all atoms was in the range of 5−10 Å for NVC and decreased slightly for the rest of the trimmed peptides (Figure S3). The percentage of helical structures per residue dropped from 43.3% in NVC to 24−27% in NVC-1, NVC-2, NVC-3, and NVC-4 trimming derivatives (Table 2). Then, it increased to 29−32% in the rest of the trimming derivatives. The molecular masses of synthesized NVC derivative peptides were verified by matrix-assisted laser desorption/ ionization time-of-flight (MALDI-TOF). Intense signals in the m/z ranges 1169−2295 Da showed the presence of successfully synthesized NVC peptides (Figure S4). We also exploited the ability of NVC peptides to be conjugated with Zn-S-5 complex to produce a stable delivery system. Interaction between NVC peptides and Zn-S-5 complex and formation of conjugate was also confirmed by MALDI-TOF (Figure S5). Theoretical molecular masses of NVC peptides can be found in Table 1. The formation of Zn-S-5 complex with molecular ion at m/z = 342 Da was confirmed by MALDI-TOF in our previous work.31 However, we found that other peaks of NVC conjugate with Zn-S-5 complex are likely C


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Figure 2. ATR−FTIR spectra of the amide I and II regions of NVC and its derivatives before and after interaction with Zn-S-5 complex. Amide I band of α-helix fingerprints can be recognized in NVC, NVC-1, and NVC-2 peptides. In the presence of Zn-S-5 complex the helicity of peptides is significantly reduced. The rest of the vibrational structures represented in the amide I band were either β-sheets or interchain interactions. Other vibrations significantly contributing to the amide II band include the side-chain of tyrosine residues. (A) NVC and NVC−Zn-S-5 complex, (B) NVC1 and NVC1−Zn-S-5 complex, (C) NVC2 and NVC2−Zn-S-5 complex, (D) NVC3 and NVC3−Zn-S-5 complex, (E) NVC4 and NVC4− Zn-S-5 complex, (F) NVC5 and NVC5−Zn-S-5 complex, (G) NVC6 and NVC6−Zn-S-5 complex, (H) NVC7 and NVC7−Zn-S-5 complex, and (I) NVC8 and NVC8−Zn-S-5 complex. The band assignments were α for α-helix, β for β-sheet and interchain interaction, and γ for tyrosine sidechain interference.

either β-sheets or interchain interactions at 1625−1630 and 1670 cm−1. Indications of α-helix structures in the amide II band at 1535 cm−1 were higher than those in amide I in almost all peptide derivatives (Table 2). Other vibrations significantly contributing to the amide II band include the side-chain of tyrosine residues at 1517 cm−1. The Zn-S-5 complex showed three major amide bands at 1570, 1600, and 1642 cm−1. The latter band is shouldered by a smaller peak at 1620 cm−1. All Zn-S-5 amide bands were lost upon interaction with NVC derivatives; however, there has been an increase in the intensity of interchain interactions at 1630 cm−1 which in most cases has already shifted from 1625 to 1630 cm−1 (Figure 2). This indicates a direct short-range interaction between the backbones of peptide and Schiff base. To investigate the structural role of NVC on toxicity, several cell viability tests were conducted. Obtained results suggest that the reduction of cell viability is dependent on applied dose, length of peptides, and presence of Zn-S-5 Schiff base. Both metastatic and malignant cell lines (PC3 and 22RV1) manifest higher sensitivity after application of NVC (Figure 3A), NVC1 (Figure 3B), NVC2 (Figure 3C), and NVC3 (Figure 3D) peptides, especially in the dose range of 8 to 62.5 μM, comparing to nonmalignant PNT1A cell line. NVC and its derivatives show strong cytotoxic effect on PC3 cells with structural and dose-dependent reduction of cell viability. However, after application of NVC−Zn-S-5 complex on PC3 cell line, viability decreases and this leads to the conclusion that complexes increase the toxicity of NVC. On the other

hand, 22RV1 cells show higher sensitivity comparing with PC3 cell line after treatment with NVC. Meanwhile, NVC increases the viability after treatment with higher concentration, which suggests that the peptide has limited anticancer properties in the case of 22RV1 cells. However, the NVC−Zn-S-5 complex increases the toxicity of NVC in the same manner like in the case of PC3 cell line. Statistically, in PC3 and PNT1A cell lines, the addition of 31 μM NVC−Zn-S-5 was significantly more toxic when compared to addition of 31 μM NVC (p < 0.05). This was observed for most cases using NVC-1, NVC-2, and NVC-3 for concentrations equal to and greater than 31 μM (p < 0.05). In contrast, the addition of NVC was significantly more toxic than NVC−Zn-S-5 in 22RV1 in most of the derivative peptides and concentrations (p < 0.05). In general, the trimmed peptides NVC1, NVC2, and NVC3 showed moderate toxicity with IC50 values from 16 μM to 62.5 μM. Overall, it is obvious that change of amphipathic character does not influence the toxicity of peptides. The removal of single Lys at position 1 (NVC1) slightly influences the toxicity comparing with NVC in the case of PC3 and 22RV1 cell lines. The elimination of further residues from hydrophilic (NVC2) and hydrophobic (NVC3) face of peptides had a more dramatic effect on the shifting of cytotoxicity activity to higher concentration. PC3 and 22RV1 cell lines showed that after treatment with NVC2 and NVC3 peptide the IC50 was found to be shifted to higher concentration. The same trend was observed after application of NVC2−Zn-S-5 and NVC3−Zn-S-5 complexes. However, D


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Figure 3. Comparison of cell viability of PC3, 22RV1, and PNT1A cell lines upon exposure to NVC peptide and its derivatives. Conjugation with Zn-S-5 significantly increased the toxic effect. Elimination of the cytotoxic activity is directly connected with losing peptide helicity. (A) NVC and NVC−Zn-S-5 complex, (B) NVC1 and NVC1−Zn-S-5 complex, (C) NVC2 and NVC2−Zn-S-5 complex, (D) NVC3 and NVC3−Zn-S-5 complex, (E) NVC4 and NVC4−Zn-S-5 complex, (F) NVC5 and NVC5−Zn-S-5 complex, (G) NVC6 and NVC6−Zn-S-5 complex, (H) NVC7 and NVC7−Zn-S-5 complex, and (I) NVC8 and NVC8−Zn-S-5 complex.

Figure 4. Investigation of the plasma membrane integrity via LDH assay. PC3 (A−C), 22RV1 (D−F), and PNT1A (G−H) cell lines were treated with peptides and peptides conjugated with Zn-S-5 complex at concentrations of 25, 50, and 100 μM. LDH activities gradually decrease from NVC1 to NVC8 indicating a helicity dependent destruction of membrane integrity. The conjugation with Zn-S-5 complex resulted in similar trends of LDH activity. Cell lines treated with NVC peptide and its derivatives are presented with blue color, whereas cell lines treated with NVC peptide and its derivatives conjugated with Zn-S-5 complex are presented with red color. E


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Figure 5. Visualization of NVC peptide and its derivative effects on the morphology of prostate cell lines. Morphological changes appear in a timedependent manner in the form of cell shrinking and blebbing in PC3, 22RV1, and PNT1A cell lines. (A) Micrographs of PC3 cell line after incubation with NVC peptide and its derivatives from 0 to 80 min, showing cytotoxic effect of NVC, NVC1, NVC2, NVC3, and NVC4 peptide and elimination of cytotoxic effect of NVC5, NVC 6, NVC7, and NVC 8. (B) As in the case of PC3 cell line, the identical cytotoxic effects are present in the 22RV1 cell line after incubation with NVC peptide and its derivatives from 0 to 80 min. (C) Micrographs of PNT1A cell line showing mild morphological changes of NVC, NVC1, NVC2, NVC3, NVC4, and NVC5 peptide after 40 min in the form of cell shrinking and without any detected effect in the case of NVC6, NVC7, and NVC8.

the Zn-S-5 complex in the case of NVC2 and NVC3 still improved the toxicity of peptides. In contrast, PNT1A cell line

exhibited a much lower sensitivity to NVC, NVC1, NVC2, and NVC3 peptides in comparison with PC3 and 22RV1 cell lines F


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Figure 6. Investigation of the structural and functional role of central glycine. NVC-G peptide has deleted central glycine, while NVC-G > I and NVC-G > K have substituted glycine with isoleucine and lysine, respectively. Cell viability of PC3 (A), 22RV1 (B), and PNT1A (C) cell lines determined by the MTT assay demonstrating the selectivity of NVC-G, NVC-G > I, and NVC-G > K for cancer cells compared with reference NVC peptide. Membrane disruption of PC3 (D), 22RV1 (E), and PNT1A (F) cell lines detected by the LDH assay demonstrating the role of central glycine in penetrating peptide function. NVC-G > I demonstrated no activity in normal cell line. Role of central glycine stability of helical structures in the NVC-G (G), NVC-G > I (H), and NVC-G > K (I) derivatives detected by FT-IR.

with detected IC50 between 47 and 125 μM. However, although Zn-S-5 complex helps peptides reduce the viability of PNT1A cell line, PNT1A is still less sensitive than PC3 and 22RV1 cell lines. Further removal of residues has led to elimination of the cytotoxic activity which can be seen in the cases of NVC4 (Figure 3E), NVC5 (Figure 3F), NVC6 (Figure 3G), NVC7 (Figure 3H), and NVC8 (Figure 3I) peptides. The sequence of the trimmed peptides NVC4, NVC5, NVC6, NVC7, and NVC8 showed gradual elimination of the cytotoxic activity as the residues were removed from the sequence, which is probably connected with loss of peptide helicity. The similar trend was obtained in the case of peptide conjugates with Zn-S-5 complex. To better understand and compare the toxicity effect of NVC peptides with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, we decided to quantify lactate dehydrogenase (LDH) as a biomarker for cell membrane damage. Obtained results show that 60 min exposure of PC3, 22RV1, and PNT1A cell lines to NVC induces their lysis (Figure 4A-I). However, it is obvious that some peptides have stronger lysis effect than the others, leading to the conclusion that lysis of peptides is strongly dependent on structure changes (trimming). PC3, 22RV1, and PNT1A cell lines treated with different concentrations of NVC peptides (25, 50, and 100 μM) resulted in similar levels of LDH release. Increasing LDH activity in the case of NVC indicates a concentration dependent destruction of membrane integrity. The LDH release for NVC at 25 μM and 50 μM was strangely lower than expected when compared to the first three trimmed peptides (Figure 4). This unusual phenomenon did

not correlate with toxicity results shown in Figure 3. However, LDH release for NVC at 100 μM concentration was correlated with toxicity results. Based on these results, we can conclude that membrane disruption by NVC was dependent on peptide concentration required to form pores, while the toxicity by NVC is dependent on mechanisms of cytotoxic action that include interaction with membranes, organelles, and proteins and other molecules. On the other hand, trimmed peptides show gradual decreases in LDH activity by removal of residues from the sequence. The highest activity was observed in NVC1 peptide and the lowest in the case of NVC8 peptide, independent of applied concentrations. The treatment of cells with NVC peptides conjugated with Zn-S-5 Schiff base resulted in a similar level of LDH activity compared with NVC alone, clearly indicating the influence of structure and concentration on LDH release. A stronger activity of NVC peptides was observed in PC3 and 22RV1 cell lines in comparison with PNT1A. Interestingly, it is noted that from NVC1 to NVC8 peptides conjugated with Zn-S-5 eliminated the LDH activity in the case of PNT1A cells. In the presence of low fractions of negatively charged elements in the membrane, the free energy of the interaction between NVC and the membrane increased, leading to decreased cytotoxicity. Similarly, cancer cells display more negatively charged elements on their membrane, thus decreasing the free energy of incorporation and increasing the cytotoxicity, as was observed in our experiment.36 However, the NVC3, NVC4, and NVC5 conjugated with Zn-S-5 showed strong loss of plasma membrane integrity observed with PC3 and 22RV1 cell lines, especially with concentrations above 50 μM. Overall, G


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Bioconjugate Chemistry LDH assay results at 100 μM treatment highly correspond with results obtained from MTT assay. This was further confirmed by the morphological assessment of cellular morphology upon NVC treatment. After treatment with NVC, NVC1, NVC2, NVC3, and NVC4, the morphology of PC3 and 22RV1 significantly changed from the 40 to 80 min point (Figure 5A,B). From the micrographs, it can be clearly seen that cells underwent morphological changes by blebbing and shrinkage. In the case of PNT1A cells it is noted that after treatment with NVC and trimmed NVC peptides, morphological changes slowly appear after 80 min in the form of cell shrinking (Figure 5C). Obviously, NVC peptides required a longer time to penetrate the cell membrane and trigger morphological change. However, further trimming of NVC peptide results in the absence of morphological changes in all cell lines indicating that NVC peptide activity is highly dependent on structure. Central Glycine Peptide Derivatives. MD simulations showed the borderline of the lost N-terminal helix precisely at the central glycine residue position (Figures S1 and S2). Three derivative peptides were used to investigate the structural and functional roles of central glycine. NVC-G peptide has deleted central glycine, while NVC-G > I and NVC-G > K have substituted Gly with Ile and Lys, respectively. The MALDITOF results confirm the molecular weight of new synthesized peptides, before and after interaction with Zn-S-5 complex (Figure S6). In detail, the observed ions at m/z = 2238.570 Da correspond to the theoretical mass of the NVC-G peptide, whereas the ions at m/z = 2351.629 Da correspond to NVC-G > I and m/z = 2366.627 Da to NVC-G > K peptide. Interaction between the peptides and Zn-S-5 was also confirmed. The mass spectrum showed formation of two molecular ions at m/z = 2238.570 Da that corresponds to NVC-G and at m/z = 2401.650 Da that corresponds to NVCG−Zn-S-5 conjugate, whereas the ions at m/z = 2351.629 Da to NVC-G > I and 2514.649 Da to NVC-G > I−Zn-S-5, 2366.627 Da to NVC-G > K and 2529.730 Da to NVC-G > K−Zn-S-5. Similarly, with data obtained from trimmed peptide derivatives and their interaction with Zn-S-5 complex we found that peaks of glycine derivatives conjugated with Zn-S-5 complex likely belong to the fragments of the complex. Viability assay showed that all peptides had consistent selectivity to cancer cell lines in contrast to normal PNT1A cell line (Figure 6A-C). A drop in viability below 80% for central glycine derivatives was observed at the following concentrations: >4 μM in PC3 cell line, >4 μM in 22RV1 cell line for NVC-G > I, >8 μM in 22RV1 cell line for NVC-G and NVC-G > K, and >8 μM in PNT1A cell line. NVC-G > I and NVC-G > K demonstrated decreased membrane penetration with reference to NVC. The latter is indicated by LDH assay (Figure 6D-F). However, NVC-G showed similar LDH activity to NVC confirming that the central glycine flexibility in the helix was not vital to penetrating peptide function. FT-IR results indicated the role of central glycine as destabilizing to the NVC peptide (Figure 6G-I). Predominant peaks in the amide I band near 1650 cm−1 showed more stable helical structures in the NVC-G and NVC-G > I derivatives, and less aggregation/β-sheet structures in the presence of Zn-S-5 (Figure 6G-I). NVC-G and NVC-G > I showed most predominant helix among the derivatives, and yet only NVCG showed LDH release potency like NVC. Exposure of PNT1A cell line to NVC-G > I did not show LDH activity; however, its toxicity was comparable to the rest of peptides.

The membrane partitioning is mostly driven by the hydrophobic nature of peptide. Amino acids such Ile or branched Val exhibit lower hydrophobicity in the lipid bilayer, which could explain why NVC-G > I had no real impact on LDH activity.37 Substitution of Gly with Ile will stabilize the helicity of peptide and reduce the positive charge enhancing the endosomal escape of NVC-G > I.38,39 NVC-G > I was among the few peptides, along with NVC-1, that displayed predominant helix in FT-IR upon complexing with Zn-S-5. The morphological state of cell lines confirmed the MTT and LDH assay results. Treatment of PC3 and 22RV1 cell lines with NVC-G, NVC-G > I, and NVC-G > K peptides results in significant morphological change within 40 min from the treatment start point (Figure 7A,B). Micrograph clearly reveals that cells are undergoing morphological changes by blebbing and shrinkage. Treatment of PNT1A cell line with NVC-G and NVC-G > K peptide revealed the same morphological changes like in PC3 and 22RV1 cell lines (Figure 7C). However, application of NVC-G > I peptide in the PNT1A cell line featuring the absence of morphological changes clearly confirming results obtained from MTT and LDH assays. Binding-Site Derivatives. An Ala scan of potential binding site residues is a standard method for functional analysis. In addition to the potential H-bonded electrostatic interactions, the aromatic functional groups of Schiff base and NVC were major candidates for direct interaction in this complex. NVC-F > A, NVC-Y > A, and NVC-H > A peptides have Phe, Tyr, and His residues substituted with Ala, respectively. NVC-AA has both Phe and Tyr substituted with Ala. The binding site of zinc-Schiff base on the peptide was studied by comparative FT-IR analysis of these derivative peptides (Figure 8). The Zn-S-5 Schiff base spectrum showed three distinct peaks near 1570, 1600, and 1642 cm−1 with a shoulder at 1620 cm−1. The loss of these peaks, visible vibrations, in the NVC−Zn-S-5 complex indicates that a different conformation of nitrogen-based hydrogen bonding was formed. Such an observation is confirmed by shifting of amide I peaks from weak, albeit long-range, hydrogen bond region (helix at 1650 cm−1) to strong hydrogen bond region (short-range interchain aggregates/sheets at 1630 cm−1). All binding-site derivatives, including NVC-H > A, showed a shift in the tip of amide I peak from 1650 to 1630 cm−1. In all tested peptides, only NVC-G > I showed a reverse shift from 1630 to 1650 cm−1 (Figure 6H), indicating weak Schiff base interaction with the peptide. Loss of tyrosine signal in amide II band (1517 cm−1) was observed in mutants lacking tyrosine (Figure 8B,C). This finding suggests an underestimation of predicted helix frequency in the amide II which was previously discussed in Table 2. FT-IR gave insight about NVC−Zn-S-5 Schiff base interactions but less insight into the role of zinc. Nuclear magnetic resonance (NMR) was used to study binding-site derivatives in more detail. The 1H NMR spectra of S-5 Schiff base was measured first (described in Experimental Section). The assignment of 1H NMR for Zn-S-5 was not as straightforward due to the complex behavior of this structure. The spectrum was used as a referencing material for further experiments (Figure S7). The 1H NMR spectra of binding-site derivatives in the presence and absence of Zn-S-5 were analyzed for signatures of Phe, Tyr, and His, with expectation that NVC complexing with Zn-S-5 will cause shifts in their aromatic side chains (Table 3).Briefly, the aromatic region of 1 H NMR spectra of NVC spanning 6.5−9.0 ppm was H


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results of the other derivatives (Figure S10). Once the Zn-S-5 complex was added to NVC peptide, a clear upfield shift of imidazole protons from His was observed in 1H NMR spectra, evidently indicating interaction of His with the Zn-S-5 complex (Figure S9A). Changes in chemical shifts in aliphatic regions of Hα were also noticed. Unfortunately, those signals were not precisely assigned and therefore could not be used as a source of additional information (Figure S9B). To verify previous observation, peptide derivatives with replaced aromatic amino acids were employed. In all cases, where terminal Phe (NVCF>A) or Tyr (NVCY>A) or both (NVCYF>AA) were substituted by Ala, the interaction of His with Zn-S-5 complex persisted. This interaction is characterized by the upfield shift of aromatic protons from the imidazole part of His (Figure S10). In the case when His was substituted by Ala in the peptide sequence (NVCH>A), no change in aromatic region was observed, confirming that His is responsible for interaction with Zn-S-5 complex. The role of His in binding of Zn-S-5 complex to NVC peptide was also confirmed by MALDI-TOF (Figure S11). Obtained peaks correspond to the theoretical mass of synthetized peptide. Similarly, with previous data, only the fragments of the complex were detected after their interaction with peptides. However, after His substitution we could not detect the presence of NVC−Zn-S-5 complex peak, which confirm the results obtained from NMR.

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DISCUSSION NVC peptide forms an α-helix structure that plays a role in its stability and contributes to its function. In peptides, helix stability is directly proportional to length due to co-operative formation and nucleation penalty.40 The α-helix is stabilized mainly by backbone hydrogen bonds and van der Waals interactions which have a favorable enthalpic contribution of ∼1 kcal/mol per residue.41 Side-chain interactions in the αhelix are strongest when similar residues are spaced in (i, i+4) order and also (i, i+3) order.40 The latter is often referred to as 3−10 helix. The π-helix involves similar (i, i+5) order but rather occurs in nature as a bulge in the α-helix (i, i+4). However, the formation of the α-helix in proteins relies on amino acid type and position, e.g., the N-terminus of a helix often involves Asn followed by Pro residue while the Cterminal is dominated by Gly.42 Certain amino acids are frequent inside the helix such as Ala, Leu, and Glu while others are less frequent such as Pro, Gly, and Asp.41 In general, Gly is known for destabilizing properties of protein conformations due to its greater flexibility than other amino acids.43 Gly and Pro are known helix breakers or benders;44 however, in some proteins, glycine can serve as notches that compact transmembrane domains due to their small size.45 Gly plays a major role in transporter proteins by gating channels via bending of α-helix structure.46 Several methods have been used to determine the role of Gly residue in the center of helices, such as using mutant derivatives that replace Gly residue,47 or using cross-species comparative analysis.48 The trimming experiment showed a pivotal role of Gly in destabilization of N-terminal helix combined with penalty of the length of that helix (Figures S1 and S2). The penalty of removing one residue at a time versus helicity was clearly overestimated in the simulation involving a single molecule, when compared to FT-IR and CD spectroscopy involving aggregates of peptides. Deletion of Gly (NVC-G) and substitution with Ile (NVC-G > I) stabilized the helix as shown in FT-IR (Figure 6G,H). The

Figure 7. Visualization of central glycine effects on the morphology of prostate cell lines. NVC-G, NVC-G > I, and NVC-G > K peptides revealed significant morphological changes on PC3, 22RV1, and PNT1A demonstrated by blebbing and shrinkage. Morphological changes are not observed in PNT1A cell line after treatment with NVC-G > I peptide. (A) PC3 cell line, (B) 22RV1 cell line, (C) PNT1A cell line.

investigated (Figure S8A and B). The assignment of aromatic region protons of Phe, Tyr, and His was straightforward due to the presence of a single residue of each of these amino acids in NVC. The accuracy of assignments was readily confirmed by I


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Figure 8. FT-IR analysis of binding-site derivatives and potential binding site. Investigation of NVC peptide binding-site integrity after substitution of aromatic residues with Ala. The interaction of Zn-S-5 complex with NVC-F > A, NVC-Y > A, NVC-H > A, and NVC-AA peptides has increased interchain conformation of hydrogen bonding. (A) NVC-F > A, (B) NVC-Y > A, (C) NVC-AA, and (D) NVC-H > A.

Table 3. 1H NMR Results Showing the Assigned Protons of Phe, Tyr, and His in the Presence and Absence of Zn-S-5 Phe peptide NVC

NVCF>A NVCY>A

NVCYF>AA NVCH>A

− 7.15 (d) 7.20 (t) 7.25 (t) − 7.20 (d) 7.23 (t) 7.29 (t) − 7.11 (d) 7.16 (t) 7.21 (t)

Tyr Zn-S-5

−

penetrating properties. It was found that Ile have the ability to easily interact with other Ile on hydrophobic surface, which leads to promotion and stabilization of peptide helicity.49 Similarly, the loss of penetration properties by disruption of helicity was reported in the case of many peptides.15,50 Our results illustrate the minor role of central helix bending in NVC toxicity and membrane penetration. In contrast, the trimming derivatives demonstrated quantitatively the roles of length and helix frequency in both cytotoxicity and membrane penetration. The cytotoxic mechanism of action for NVC is associated with peptide binding, followed by the lysis of cell membrane or localized disruption or pore formation. Peptide modes of action are strongly governed by the chemical nature of peptide and its structure.51,52 In most cases, the cell membrane is the primary target of cationic peptides, which can target cancer cells with minimal toxicity to normal human cells. As will be discussed, the differences between normal and cancer cell membranes provide some selectivity, yet at a narrow therapeutic range. The selectivity of the peptide to cancer vs normal tissue is beyond the scope of this work. Here, we describe optimization of the NVC shuttle prior to its conjugation with proper homing agent (e.g., homing peptide) for selective targeting. To investigate the cytotoxic effect of NVC peptides we focused our interest on the peptide’s chemical/structural properties, which correlated with MTT and LDH assay results. Our results revealed a decrease in cytotoxicity, as the trimming of NVC peptide was preceded in PC3, 22RV1, and PNT1A cell lines. Cutting of the residues from the N-terminus affects the loss of the essential structural properties of NVC peptide for the interaction and penetration of lipid bilayers. The loss of peptide penetrating activity is sought in the disruption of helicity.53,54 Along these arguments, it has also been reported that penetration of membrane bilayer

His Zn-S-5

7.15 (d) 6.71 (d) 6.71 (d) 7.20 (t) 6.98 (d) 6.98 (d) 7.25 (t) − 6.75 (d) 6.75 (d) 7.06 (d) 7.06 (d) 7.20 (d) − − 7.23 (t) 7.29 (t) − − −

−

Zn-S-5

7.17 (s) 7.06 (s) 8.54 (s) 7.96 (s) 7.18 8.55 7.18 8.55

(s) (s) (s) (s)

7.03 7.96 7.07 7.96

(s) (s) (s) (s)

7.13 (s) 7.00 (s) 8.50 (s) 7.91 (s) 7.11 (d) 6.66 (d) 6.66 (d) − − 7.16 (t) 6.94 (d) 6.94 (d) 7.21 (t)

central glycine derivatives retained toxicity and membrane penetration properties, demonstrated mostly by NVC-G, yet NVC-G > I showed interesting selectivity and less toxicity in normal cells (Figure 6). The central glycine is situated on the side of the positively charged amino acids in the helix. Replacing glycine with isoleucine influenced the symmetrical segregation of hydrophobic and hydrophilic residues in NVC. On the other hand, replacing glycine with lysine agreed with NVC’s symmetry. A recent study reported that loss of helicity diminishes the cytotoxic effect of G(IIKK)3-NH2 peptide. Elimination of N-terminal Gly leads to reduction of peptide helicity and loss of penetrating properties due to reduction of the binding ability to the phospholipid layer. On the other hand, the addition of Ile to C-terminal improved the helical conformation of (IIKK)3INH2 peptide and increased the J


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well-known fact that the tetrahedral binding geometry of free zinc(II) ions often induces helical stability and peptide dimerization.57 These explain the higher toxicity of NVC peptides modified with Zn-S-5 complexes. The interpretation of FT-IR results in binding of Zn-S-5 to NVC, particularly the amide I region, supports the role of both helical stability (persistent peak at 1650 cm−1) and short-range H-bonds resulting from interchain amides of both NVC and S-5 backbone (induced peak at 1630 cm−1 upon addition of Zn-S5) in membrane disruption and toxicity. Of all the peptide variants, the only exception to this phenomenon was NVC-G > I (Figure 6H) which did not show an induced peak at 1630 cm−1. The stabilization of peptide and Zn(II) ion across the membrane thus required a carrier with limited competition of other molecules from zinc(II) ions, hence the Schiff base. Previous studies emphasized the role of metal ion coordinated to Schiff base in boosting cytotoxicity and selectivity to the cancer cell lines.24−26 A very important aspect was to identify and characterize the binding between Zn-Schiff base and NVC. In our last work, we had assumed that the Schiff base interacted with the aromatic residues of the NVC C-terminus. Electrochemistry showed that zinc was accessible and able to target the carboxylic group of C-terminus.31 To our surprise, the NVC−Zn-S-5 complex was stable after amidation of Cterminus of NVC (C-terminus amidation is commonly used to stabilize peptides in biological settings). NVC contains one His residue that can directly interact with zinc(II) ions. The phenomena of free Zn(II) ions binding to peptides are naturally mediated by directly coordinating Cys or His ligands and indirectly through water molecules. The tetrahedral binding geometry of free zinc(II) ions often induces helical stability and peptide dimerization.48,57,58 This arena is open for future development of NVC to control zinc(II) ion loading capacity. On the other hand, the Schiff base is rich in nitrogen donors and/or acceptors and aromatic rings are capable of coordinated bonding with ions and peptide chains. It was previously chosen as a coordinated system that can be dissociated from NVC intracellularly. The high flexibility of the Schiff base structure was a limiting factor for deep study via CD spectroscopy and NMR. Nevertheless, strong coordination bonding was observed via FT-IR between the organic components of the NVC−Zn-S-5 complex. Three aromatic residues of NVC, namely Phe, Tyr, and His, were investigated for their role in binding to Zn-S-5 via NMR (Table 3) and MALDI-TOF (Figure S11). The results presented here, combined with previous electrochemistry findings,31 can be used to establish a better theoretical model of NVC−Zn-S-5 complex. The dynamic and flexible nature of the peptide and Schiff base structures was indicated by various methods, and strong NVC/S-5 intrachain H-bond interactions were clear in FT-IR. On the other hand, NMR and MALDI-TOF show direct NVC/Zn interaction through His residue either directly or through water molecule(s). In this study, defining the critical NVC residues in this complex was a first step in exploring this Zn(II) ion delivery system. Trimming and central glycine peptide derivatives explored the roles of peptide helicity in membrane-penetration and toxicity. We have also identified His as the sole Zn-Schiff base binding site among aromatic residues. Finally, the progress in this field can be seen in many dimensions: first, understanding the contribution of each component of this complex to toxicity; second, engineering NVC selectivity and

by peptides is highly dependent on concentration of peptides to lipids.30 Disintegration of anionic lipid membrane by NVC takes full effect at peptide to lipid ratio ∼1:10, where the peptide is oriented parallel to the membrane surface as αhelical. It is observed that at peptide to lipid ratio ∼1:100, membrane showed starting leakage of calcein fluorescent dye from calcein-filled vesicles, and as the concentration of peptide increased, the membrane defects were more severe. Hydrophobic residues of NVC were found to be at minimum distances from the center of the dodecylphosphocholine micelle at ratio ∼1:15 and lower, leading to the formation of transient pores or membrane transient disruptions. However, it is very interesting that in ratio range between ∼1:15 and ∼1:100, no evidence of peptide reorientation along the bilayer was found. These observations clearly demonstrate the effect of concentration on the penetrating properties of NVC and its impact on integrity of anionic lipid membranes. Other comparative studies on the NVC were also reported.29,55 Similarly, our results revealed that the cytotoxic effect of trimmed peptides is highly dependent on applied concentration and membrane composition. The morphological features of PC3, 22RV1, and PNT1A cell membranes support arguments that cell death was induced by disruption of membrane integrity. Reduction of cell membrane stability seen under the microscope correlates with results obtained by MTT and LDH assays. However, PNT1A cell line shows better membrane stability resistance to NVC peptides compared with that of PC3 and 22RV1 cell lines. Explanation can be found in the size of the peptide and the cell membrane and membrane structure. If we assume that NVC and NVC trimmed peptides have a perfect α-helix conformation, the distance between Cand N-termini would decrease from approximately 2.9 nm (NVC) to 1.7 nm (NVC8). Taking into consideration that membrane thickness is >4 nm, while the NVC in its standard helical form is approximately 2.9 nm long, we believe that the shortest/trimmed NVC will require double the quantities to cover the hydrophobic region and form the pore.56 Also, we should take into consideration that trimming of NVC will lead to a decrease of net charge, which can limit the interaction between negatively charged elements on the membrane and the peptide. This will result in an increase of free energy of interaction between peptide and membrane, which leads to a decrease of peptide toxicity.27,36 On the other hand, it is also reported that NVC has low cytolytic activity in the presence of zwitterionic membrane, which can be a reason for longer internalization of NVC in the case of PNT1A cells.27 This confirms our previous conclusion, that the degree of membrane disruption is dependent on peptide structure, concentrations, and membrane structure. In order to address the effect of Zn-S-5 complex to NVC, it is important to study the changes occurring in the peptide structure and their role in toxicity and membrane penetration. Conjugation of Zn-S-5 complex to NVC peptides improves the toxicity of applied peptide, especially at lower applied concentration. Previously, we proved that Zn-S-5 has a toxic effect at higher concentration and that oxidative stress is not the main cause of cell death, which excludes the possibility that Zn-S-5 has the main role in toxicity.31 We found that Zn-S-5 has the ability to mimic the function of zinc ion transporters to increase the intracellular free zinc ions, which could further induce antitumor apoptotic effects. These arguments confirm that the loss of peptide penetrating properties and toxicity through trimming was the result of the loss of helicity. It is a K


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diethylenetriamine (1080 μL) under stirring and heating under reflux in methanol (35 mL) for 6 h. Color turned to orange. After cooling, methanol was added to 50 mL. Zinc(II) perchlorate hexahydrate (0.372 g) was dissolved in 50 mL of water and Schiff base S (5 mL) was added with stirring to obtain Zn−S Schiff base complex. Light orange solution was heated at 80 °C for 2 h. Solution was then filtrated and water was added to reach 100 mL. Synthesis of NVC and Its Derivatives. The synthesis of NVC peptides was performed by Liberty Blue peptide synthesizer (CEM, Matthews, NC, USA), by solid phase approach using standard Fmoc methodology in a reaction vessel. The 4-methylbenzhydrylamine resin was used as first building block. Deblocking of Fmoc protecting group was performed with 20% piperidine (v/v) in 80% N,Ndimethylformamide. Each coupling reaction was accomplished by using N,N,N′,N′-tetramethyl-O-(1H-benzotriazol-1-yl)-uroniumhexafluorophosphate as activator and N,N-diisopropylethylamine as activator base. The cleavage of side chain protecting groups and resin was performed by treating the peptides resin with 95% trifluoroacetic acid (TFA, v/v), 2.5% H2O (v/v), and 2.5% triisopropylpropylsilane (v/s) for 30 min at 38 °C under microwave irradiation. The isolation of the peptide was performed by precipitation with cold (−20 °C) diethyl ether followed by centrifugation (30 mm ⌀, 6000 rpm, 3 min) and lyophilization. Sequences of all synthetized peptides can be found in Table 4. NVC Modification by Zn-S-5 Schiff Base. A stock solution of NVC peptide (1 mM) was mixed with Zn-S-5 (1 mM) at a 1:1 ratio in ACS water. The sample was incubated overnight at room temperature using PTR-60 360° MultiFunction Rotator (VWR Singapore Ltd.). Finally, the NVC was filtered using Amicon Ultra 0.5 mL centrifugal filters (Merck Millipore, Billerica, MA, USA) to remove unbound residual impurities. Peptide Structure and MD. Peptide structures were built using UCSF Chimera v 1.10.2.59 The standard α-helix (φ = −57°, ψ = −47°) was the choice for all starting structures using the Dunbrack rotamer library. Protonation states at pH = 7.4 were estimated using H++ server assuming 0.15 mM salinity and default dielectric constants.60 Coordinate and topology files constructed by H++ server for implicit solvent (using Amber ff14SB force field) were directly used in MD simulations. The generalized Born model for implicit solvent is among the most widely adapted methods for MD simulations. A recently improved model that has better agreement in secondary structure preferences with explicit solvent MD is employed in this work.61 Using the Sander module of Amber 14, structures were minimized by 2500 steepest-descent steps and equilibrated to 37 °C for 25,000 steps (1 fs). Eight rounds of MD simulations, encompassing 50 ns each, were performed in steps of 2 fs. A nonbonded cutoff of 16 Å was used. Simulations were performed at the Metacentrum grid (Czech Republic) which is managed by PBSPro scheduler system. Hardware choice was based on infiniband connections and parallel computing via message passing interface (MPI). Each simulation utilized 10 CPUs and 8 Gb RAM memory. Each CPU was 2× 12-core Intel Xeon E52650v4 (2.20 GHz). Analysis of trajectories was performed using cpptraj module in Amber 14 and using the Bio3D module in R language.62 VMD was used for visualization of structures.63 Secondary structure analysis was done according to standard DSSP method.64

potency to transform it from a pore-forming peptide to a transport shuttle; third, understanding and control of the zinc(II) ion loading capacity and release process.

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CONCLUSION This paper describes in the first step an iterative process aimed at the design of a more potent peptide for drug delivery. The trimming of NVC suggest the functional roles of key amino acids in the helicity of derivatives and toxicity. The trimming of the N-terminal residues significantly stabilized the helicity of NVC1 and NVC2 derivate, strongly increasing the toxicity. However, further destabilization the N-terminal helix without affecting the C-terminal helix structure leads to disruption of derivative helicity and decrease of toxicity. Further, the proposed central glycine derivatives demonstrate that substitution of glycine stabilized the helicity with a minor role in cytotoxicity clearly showing the role of length and helix frequency in both toxicity and membrane penetration. Interestingly, the use of simple chelating zinc Schiff base complex (Zn-S-5) conjugate with His ligands through water molecules stabilized the helicity of NVC. The complexes increase the free energy of interaction between NVC and the membrane leading to the decrease of the cytotoxicity and LDH activity in the case of PNT1A cells. Furthermore, we suggest Zn-S-5-NVC conjugates can behave as artificial metallochaperones because they have the potential to deliver metal ions into the cell and to mimic the function of zinc ion transporters to increase the intracellular free zinc ions, which could further induce antitumor apoptotic effects. These arguments are confirmed by preserving of NVC toxicity regardless of peptide trimming and loss of helicity.

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EXPERIMENTAL SECTION Chemicals and Reagents. The chemicals and solvents were supplied by Sigma-Aldrich (St. Louis, MO, USA), in ACS purity, used without further purification. Synthesis of Schiff Base (S) and Schiff Base Complex Zn-S-5. Synthesis of Schiff base and Schiff base complex (Scheme 1) was conducted following protocol used in our previous work.31 Schiff base [(2-[(E)-2-pyridylmethyleneamino]-N-[2-[(E)-2-pyridylmethylene-amino]ethyl]ethanamine)] was prepared mixing 2-pyridinecarboxaldehyde (1902 μL) and Scheme 1. Schematic Representation of Schiff Base (S-5), and Zinc(II) Ion Coordination to Schiff Base (Zn-S-5)

L


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Bioconjugate Chemistry Table 4. Sequences of NVC Peptides with Theoretical Calculated Properties name

sequence

total charge (pH 7)

pI of helix

hydrophobicity (%)

molecular weight

NVC NVC1 NVC2 NVC3 NVC4 NVC5 NVC6 NVC7 NVC8 NVCF>A NVCY>A NVCH>A NVCYF>AA NVCΔG NVCG>I NVCG>K

KNLRRIIRKGIHIIKKYF NLRRIIRKGIHIIKKYF LRRIIRKGIHIIKKYF RRIIRKGIHIIKKYF RIIRKGIHIIKKYF IIRKGIHIIKKYF IRKGIHIIKKYF RKGIHIIKKYF KGIHIIKKYF KNLRRIIRKGIHIIKKYA KNLRRIIRKGIHIIKKAF KNLRRIIRKGIAIIKKYF KNLRRIIRKGIHIIKKAA KNLRRIIRKIHIIKKYF KNLRRIIRKIIHIIKKYF KNLRRIIRKKIHIIKKYF

7.1 6.1 6.1 6.1 5.1 4.1 4.1 4.1 3.1 7.1 7.1 7 7.1 7.1 7.1 8.1

11.94 11.91 11.91 11.91 11.52 11.02 11.02 11 10.55 11.94 12.45 11.94 12.45 11.94 11.94 11.96

37.83 37.21 36.12 34.11 34.55 32.52 29.02 27.12 28.39 31.68 34.91 36.60 29.63 41.16 45.96 40.24

2296.88 2168.71 2054.60 1941.44 1785.26 1629.07 1515.91 1402.75 1246.56 2220.78 2204.78 2230.82 2128.68 2239.83 2352.99 2368.00

NMR Spectroscopy. 1H NMR spectra were measured on JEOL ECA-500 at 25 °C in D2O with Watergate pulse sequence for elimination of solvent signal. NMR spectra are reported as chemical shifts in parts per million (ppm, δ). The splitting patterns are reported as s (singlet), d (doublet), and t (triplet). The assignment of 1H NMR (500 MHz, D2O) for S-5 Schiff base was as follows: δ 2.52−2.58 (m, 1H), 2.81 (t, J = 6.4 Hz, 2H), 3.00−3.02 (m, 1H), 3.07−3.09 (m, 1H), 3.24−3.25 (m, 2H), 3.52−3.54 (m, 1H), 7.18−7.20 (m, 1H), 7.37 (d, J = 3.8 Hz, 2H), 7.59−7.61 (m, 2H), 7.70−7.80 (m, 2H), 7.90 (s, 1H, CHN), 8.26 (d, J = 4.0 Hz, 1H), 8.42 (d, J = 3.6 Hz, 1H). Determination of NVC Cytotoxicity - MTT Assay. MTT assay was conducted on three human prostate cell line PC3 (grade IV prostate cancer bone metastasis), 22RV1 (androgenresponsive human prostate carcinoma), and PNT1A (normal prostate epithelium). Briefly, the suspension of 5000 cells in 50 μL medium was added to each well of microtiter plates, followed by incubation for 24 h at 37 °C with 5% CO2 to ensure cell growth. After 24 h treatment (medium containing 0.5−500 μM NVC), 10 μL of MTT [5 mg/mL in phosphate buffered saline (PBS)] was added to the cells and the mixture was incubated for further 4 h at 37 °C. After that, MTTcontaining medium was replaced with 100 μL of 99.9% dimethyl sulfoxide, incubated for 5 min, and the absorbance of the samples at 570 nm was determined using Infinite 200 PRO (Tecan, Maennedorf, Switzerland). Statistical analysis was performed by Student’s t test for independent samples. A pvalue below 0.05 was considered significant. Determination of LDH Release (LDH Assay). Three human prostate cell lines PC3, PNT1A, and 22RV1 were treated with NVC and NVC derivatives conjugated with Zn-S5 complex at concentration of 25, 50, and 100 μM. After 1 h of treatment, cells were stained using the Pierce LDH Cytotoxicity Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s protocol and analyzed by using Infinite 200 PRO (Tecan). Evaluation of Effect of NVC on Cellular Morphology. The change of morphology was followed by the microscope Invitrogen EVOS FL Auto Cell Imaging System (Thermo Fisher Scientific, Waltham, MA, USA). Briefly, the suspension of 10,000 cells/well in six-well dishes, followed by incubation for 24 h at 37 °C with 5% CO2 to ensure cell growth. Just

Matrix-Assisted Laser Desorption/Ionization Time-ofFlight (MALDI-TOF) Analysis of NVC and NVC−Zn-S-5 Complex. The mass spectra were acquired on a MALDI-TOF MS Bruker UltrafleXtreme (Bruker Daltonik GmbH, Bremen, Germany). The instrument was controlled by the flexControl v 3.4 and flexAnalysis v 3.4 software. The 2,5-dihydroxybenzoic acid was used as MALDI-TOF matrix (Bruker Daltonik GmbH). The saturated matrix solution was prepared in 30% acetonitrile and 0.1% TFA. All measurements were performed in reflector positive mode in the m/z range 0−4 kDa. The mass spectra were typically acquired by averaging 2000 subspectra from a total of 2000 laser shots per spot. Laser power was set 5−10% above the threshold. The calibration was done using standard peptide calibration mixture obtained from Bruker (Bruker Daltonik GmbH, Bremen, Germany). CD Spectroscopy. CD experiments were performed on a Jasco J-715 spectropolarimeter at 298 K in a 0.02, 0.05, and 1 cm quartz cells. The spectral range was 200−800 nm. The concentrations of ligand and complexes solution were set at 3 mM. The spectra are expressed in terms of Δε = εl − εr, where εl and εr are molar absorption coefficients (in M−1 cm−1) for left and right circularly polarized light, respectively. Figures present spectra in spectropolarimeter unit’s Δθ [mdeg]. Secondary structure analysis was performed by graph visualization, and also by knowledge-based approaches, namely, K2D234 and K2D3.35 Attenuated Total Reflectance FT-IR. FT-IR spectra were collected using a Nicolet iS10 FT-IR spectrometer with attenuated total reflection attachment (Thermo Fisher Scientific, USA), equipped with diamond crystal. The recording of spectra was done at 25 °C from 4000 to 650 cm−1 at a resolution of 2 cm−1. Each spectrum was acquired by merging 128 interferograms. Peptide samples were directly analyzed in lyophilized form. Curve fitting was performed on MagicPlot Student 2.7.2 (Magicplot Systems, LLC, Russia). Tails of neighboring bands were subtracted before curve fitting. Each spectrum (spanning the amide I and amide II bands) was fitted with no more than 8 curves to obtain an adjusted R2 value of 0.999. Limits for data processing were set at 10,000 iterations and 1.0 × 10−16 minimum deviation of residual sum of squares. Assignments of wavenumbers were as follows: 1650−1660 cm−1 for α-helix, 1670 and 1630 cm−1 for β-sheets and interchain aggregations. M


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before imaging, the cells were washed carefully with PBS to remove any detached or floating cells in the medium. After treatment by NVC peptides in concentration of 100 μM, the cells were photographed every 5 min for 90 min.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.bioconjchem.8b00370. Time evolution of secondary structural changes; Percentage of secondary structural distribution during MD simulation of trimmed peptide derivatives; RMSD for all atoms during MD simulation of trimmed peptide derivatives; MALDI-TOF mass spectra; 1H NMR spectra; MALDI-TOF mass spectra (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel.: +420-5-4513-3350. Fax: +420-5-4521-2044. ORCID

Roman Guran: 0000-0002-2912-714X Vojtech Adam: 0000-0002-8527-286X Author Contributions

V.M. contributed to design of experiment and writing of manuscript, synthesis of Schiff-base, and synthesis of NVC peptides. Y.H. contributed to writing of manuscript and MD simulation. H.B. contributed to the cultivation of tissue culture, MTT assay, LDH assay and microscopy testing; R.G. contributed to the MALDI-TOF analysis. T.P. contributed to the NMR analysis. K.S.S. contributed to the CD analysis. L.R. contributed to the ATR-FTIR analysis. Z.H., A.M., and P.K. contributed to reagents/materials/tools and revision of manuscript, and V.A. was principle investigator and contributor to design and infrastructure of work. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work is financially supported by The Czech Science Foundation (GACR 17-12816S), IGA grant, no. TP_4/2017 and CEITEC 2020 (LQ1601) and with financial support from the Ministry of Education, Youth and Sports of the Czech Republic under the National Sustainability Programme II. Computational resources were provided by the CESNET LM2015042 and the CERIT Scientific Cloud LM2015085, provided under the programme “Projects of Large Research, Development, and Innovations Infrastructures”. T.P. thanks to grant No. LO1204 (Sustainable development of research in the Centre of the Region Haná) from the National Program of Sustainability I, MEYS, Czech Republic.

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