Antimicrobial Activity of Chimera Peptides Composed of Human

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Antimicrobial activities of chimera peptides composed of human neutrophil peptide 1 (HNP-1) truncated analogues and bovine lactoferrampin Natalia Ptaszy#ska, Katarzyna Gucwa, Anna ##gowska, Dawid Debowski, Agata Gitlin-Domagalska, Jan Lica, Mateusz Heldt, Dorota Martynow, Mateusz Olszewski, S#awomir Milewski, Tzi Bun Ng, and Krzysztof Rolka Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.8b00440 • Publication Date (Web): 26 Jul 2018 Downloaded from http://pubs.acs.org on July 27, 2018

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Bioconjugate Chemistry

Fig. 1. CD spectra of chimera peptides CH8, CH9, CH10 and their constituent peptides recorded in H2O (A – C) and trifluoroethanol (D – F). The concentration of all studied peptides was 18 µM. 345x142mm (300 x 300 DPI)

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Fig. 2. Dose-response viability curves for A549 and U-2 OS cell lines. Cells were seeded at 96-well plates at 1.5 × 103 and exposed to the action of CH9 for 72 h. Results obtained with MTT assay. 20x6mm (600 x 600 DPI)


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Fig. 3. Comparison of cytotoxicity effect of CH8 on primitive and standard cell cultures. 20x8mm (600 x 600 DPI)



Fig. 4. ROS generation. HEK 293 CH8 and CH9 chimeras were added in the concentration equal to 150 µM. HL-60 cell line cells were treated with the concentrations 25 µM for CH8 and 50 µM for CH9 and CH10. Positive control was tested with 250 µM of H2O2. A panel two-parameter dot plot (7-AAD – apoptosis level, CM-H2DCFDA oxidisation -ROS level both in log scale). B ROS levels for both cell lines in comparison to controls. 211x152mm (300 x 300 DPI)


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Fig. 5. Fluorescence microscope images of CH10-CF and its constituents (CF-LFampB-C and HNP[Cys5]- CF) uptake by S. epidermidis. Cells were grown to the mid-log phase and exposed to the action of the compounds at concentration 200 µM for 30 min. 205x172mm (150 x 150 DPI)



Fig. 6. Intermolecular disulfide bridge formation in CH10. The reaction progress was monitored by HPLC and MS. Modification of LFampB-C by 2,2’dithiodipyridine at 0 min (A), and 60 min (B) and (F), reaction of activated LFampB-C and HNP[Cys5] after 60 min incubation (D) and (G), purified CH10 (E). 23x19mm (600 x 600 DPI)


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Antimicrobial activities of chimera peptides composed of human neutrophil peptide 1 (HNP-1) truncated analogues and bovine lactoferrampin Natalia Ptaszyńska†*, Katarzyna Gucwa†,‡, Anna Łęgowska†, Dawid Dębowski†, Agata GitlinDomagalska†, Jan Lica‡, Mateusz Heldt‡, Dorota Martynow‡, Mateusz Olszewski‡, Sławomir Milewski‡, Tzi Bun Ng§, Krzysztof Rolka† †

Department of Molecular Biochemistry, Faculty of Chemistry, University of Gdańsk, Wita

Stwosza 63, 80-308 Gdańsk, Poland ‡

Department of Pharmaceutical Technology and Biochemistry, Faculty of Chemistry, Gdańsk

University of Technology, Narutowicza 11/12, 80-233 Gdańsk, Poland §

School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, New Territories, 99-999 Hong Kong, China *corresponding autor, email: [email protected], tel: +48 58 523 50 92, fax: +48 58 523 50 12

Abstract Three chimera peptides composed of bovine lactoferrampin and the analogue of truncated human neutrophil peptide 1 were synthesized by the solid-phase method. In two compounds peptide chains were connected via isopeptide bond whereas in the third one disulfide bridge served as a linker. All three chimeras displayed significantly higher antimicrobial activity than the constituent peptides as well as their equimolar mixtures. The one with a disulfide bridge displayed selectivity towards Gram-positive bacteria and was able to penetrate bacterial cells. The chimeric peptides demonstrated low in vitro mammalian cytotoxicity, especially against benign cells. The significance of linker type was also reflected in the secondary structure and proteolytic stability of studied compounds. Presented results proved that such chimeras are good lead structures for designing antimicrobial drugs. Introduction Antibiotics are reckoned as one of the greatest medical achievements of the twentieth century. Unfortunately, as a result of their common (and often excessive) use in medical practice, many pathogenic bacteria and fungi have become resistant to most of commercially available antibiotics. In turn, it contributed to a major global health issue associated with life-



threatening infections, that are becoming harder to treat with currently accessible antibiotics. Thus, the development of new, effective antibiotics is one of the biggest challenges of the medicinal chemistry in the twenty-first century. In recent years, a growing interest in antimicrobial peptides (AMPs) was observed. AMPs constitute an important element of the body's natural immunity against microorganisms. They are widespread in nature and were extracted from various organisms such as plants, insects, amphibians, humans and even bacteria. Over 2,500 of AMPs have been deposited in the Antimicrobial Peptide Database (http://aps.unmc.edu/AP/main.php) up to date. AMPs activity is generally associated with their interaction and, in the next step, disruption of bacterial cell membranes.1 There are several reasons to consider AMPs as antibiotics: they are relatively small, easy-to-synthesize peptides, exhibiting a broad spectrum of antimicrobial activity, and low tendency to the drug resistance development.2 However, as several recent studies have shown, resistance to AMPs can in fact evolve at high rates (at least in vitro), generating mutants with, sometimes high, levels of resistance.3,4 The application of AMPs as antibiotics meets some obstacles such as susceptibility towards proteolytic degradation, moderate activity, toxicity against eukaryotic cells and the potential immunogenicity. These unwanted consequences of therapeutic use could drastically undermine innate immune system's ability to control and fight microbial infections and may lead to development of allergies.2 Lactoferrin (LF), 80 kDa iron-binding glycoprotein, identified in 1939 in bovine and later in human milk, is an important element of the innate immune system. LF is one of the major proteins in all exocrine secretions and is involved in various physiological functions, but - its broad-spectrum antimicrobial activity seems to be the most important feature.5 Hence, LF was extensively studied for its possible application in the medicinal practice.6 Moreover, this protein upon proteolytic cleavage of the polypeptide chain, is a rich source of AMPs. The most studied are: LF(1–11), lactoferricin (LFcin) and lactoferrampin (LFamp). The first one is an N-terminal fragment of human LF7, whereas the remaining two are 17-418 and 268-284 fragments (or their slightly shorter or longer variants) of the N1 domain of bovine LF.9 All these peptides are positively charged under physiological conditions. The last one (LFamp), regardless sequence differences (isolated from various species) displays an amphipathic character adopting an α-helix structure of the N-terminal fragment under conditions that mimic interaction with cell membranes10, which is considered to be responsible for its membrane-mediated activities.9 Bovine LFamp exhibits broad-spectrum bactericidal activities against a range of Gram-positive and Gram-negative bacteria, yeast and parasites. In contrary,


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its human variant is significantly less active.11 In 2008 Lundy et al.12 designed a synthetic AMP, which displayed a broad spectrum of antibacterial activity. The peptide, named 2Abz23S29, derived from the C-terminal fragment of the human neutrophil peptide 1 (HNP-1), comprised of its modified (Cys19→Cys(Acm), Gly23→2Abz, Cys29→Ser) middle 15 – 29 fragment (see Table 1). This linear peptide exhibited antimicrobial activity similar to that of the parent HNP-1. Low-molecular mass antimicrobial compounds (conventional antibiotics) possess limited number of functional groups, which could be subjected to modification or utilized for conjugation with other molecules. In addition, often they are crucial for biological activities and therefore cannot be replaced. In this respect AMPs are more inclined for modifications, expanding possibility to design antimicrobial agents against bacteria resistant to conventional antibiotics. One of the promising approaches was reported in 2009 by Bolscher et al.13 They synthesized a hybrid peptide, named LFchimera composed of LFcinB(17-30) and LFampinB(265-284). The first peptide was attached by the isopeptide bond formed (through ε-NH2 group) by its C-terminal Lys residue to the C-terminus of the second one. The obtained compound displayed bactericidal activity (against both Gram-positive and Gram-negative bacteria) stronger than constituent peptides or their equimolar mixture, including clinical isolates of antibiotic-resistant Staphylococcus aureus and Escherichia coli.14 The authors showed that LFchimera possessed also antifungal15 and antibiofilm activities, reducing viability of multispecies bacteria in biofilm better than antiseptics used in the medicinal practice.16 Taking the above mentioned published results into consideration, we decided to design and synthesize series of peptidic hybrids composed of 2Abz23S29 analogues (named here as HNP) and bovine LFampB. Primary structures of synthesized chimeras and their constituent peptides are shown in Table 1. Two different covalent linkers were used: (i) isopeptide bond, considered as non-cleavable in biological systems and (ii) redox-sensitive disulfide bridge. The first chimera (CH8) is composed of LFampB, which is coupled through the isopeptide bond formed by its C-terminal carboxyl group to the reference peptide HNP by the ε-amino group of the C-terminal Lys. In the second chimera (CH9), Cys(Acm) residue of HNP was replaced by L-α-aminobutyric acid (Abu), which is often used to substitute sulfur containing amino acids (Met or Cys). In the third chimera (CH10), constituent peptides were coupled by the disulfide bridge formed by the thiol groups of Cys5 of HNP and the Cys residue attached at the C-terminus of LFampB, respectively. In all three chimeras, C-terminal carboxyl groups



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not involved in formation of intermolecular amide bonds, were converted to amide groups. In order to determine the cellular uptake of CH10, its fluorescently-labelled analogue (CH10CF) and constituent peptides (HNP[Cys5]-CF and CF-LFampB-C) were also synthesized. Table 1. Primary structures and HPLC and MS analyses of synthesized chimeras, constituent peptides and their precursors. Chimera/ Constituent peptide

CH8 HNP-K-LFampB

Primary structure

Calculated MW [Da] (m/z)a

RYGTC(Acm)IYQ(2Abz)RLWAFSK-NH2

4110.8 (4111.7)

tR[min]

15.68

WKLLSKAQEKFGKNKSR-

CH9 HNP[Abu5]-K-LFampB CH10 HNP[Cys5]-LFampB-C CH10-F HNP[Cys5]-CFLFampB-C HNP1 HNP1 (15-29) HNP(2Abz23S29) HNP-K HNP[Abu5]- K HNP[Cys5]

HNP[Cys5]-CF

LFampB LFampB-C CF-LFampB-C

RYGTAbuIYQ(2Abz)RLWAFSK-NH2 WKLLSKAQEKFGKNKSR-

RYGTCIYQ(2Abz)RLWAFS-NH2 WKLLSKAQEKFGKNKSRC-NH2

RYGTCIYQ(2Abz)RLWAFSK-NH2 CF WKLLSKAQEKFGKNKSRC-NH2

4021.2 (4023.3)

16.16

4030.0 (4030.9)

17.33

19.88 4516.5 (4517.4)

RYGTC5IYQG9RLWAFC15 RYGTC(Acm)IYQ(2Abz)RLWAFS-NH2 RYGTC(Acm)IYQ(2Abz)RLWAFSK-NH2 2081.4 (2083.1) RYGTAbuIYQ(2Abz)RLWAFSK-NH2 1991.8 (1993.6) RYGTCIYQ(2Abz)RLWAFS-NH2 1881.4 (1883.1) 2367.9 RYGTCIYQ(2Abz)RLWAFSK-NH2 (2368.1) CF WKLLSKAQEKFGKNKSR-NH2 WKLLSKAQEKFGKNKSRC-NH2 CF-WKLLSKAQEKFGKNKSRC-NH2

2047.4 (2048.9) 2150.6 (2152.5) 2508.9 (2510.9)

19.65 18.64 18.57 21.15

20.22 12.94 18.50

where CF is 5(6)-carboxyfluorescein, MW is molecular weight, tR is retention time a

Molecular weights of the peptides were determined on a Bruker Briflex III MALDI-TOF spectrometer (Bruker, Geremany). The average values are given.


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Results and discussion

Peptide synthesis Constituent and chimera peptides were synthesized by the solid-phase method. In case of CH8 and CH9, 4-methyltrityl (Mtt) group was applied to protect ε-amino group of Lys residue attached at the C-terminus of HNP and [Abu5]HNP. Its selective cleavage allowed the assembly of the second peptide chain (LFampB) to the peptidyl-resin. The same protecting group was also used for the synthesis of fluorescent-labelled compounds. Intermolecular disulfide bridge, serving as a linker in CH10, was formed selectively in solution between unprotected constituent peptides. The thiol function of LFampB-C was activated by 2,2’dithiodipyridine. MS and HPLC analyses of synthesized compounds are shown in Table 1.

Peptide stability study in human serum The results of experiments show that all three chimeras and their peptide components are not resistant to proteolytic activity of enzymes present in human serum. The most stable was chimera CH8. It remained intact after first hour of incubation in human serum, whereas 68% of the peptide was still present after 2 hours (see Fig. S1 in supporting information). Only 7% of CH9 was degraded in the first hour of incubation, however, after two hours only 23% of the initial compound was detected. After 24 hours, peaks corresponding to both chimeras vanished completely. The constituent peptides of above mentioned chimeras, HNPK, HNP[Abu5]-K and LFampB, were significantly less stable. In case of first two compounds, after 1 h of incubation 10% and 40% of peptides remained intact respectively. After next hour of incubation they were practically degraded. LFampB manifested much higher stability and upon 2 h incubation the amount of intact peptide was about 55 % of its initial concentration. It is worth to notice that for all peptides studied, the proteolysis was completed after 24 hours. Due to the overlapping of new appearing peaks (coming from both, studied compounds and serum), we were not able to determine products of degradation. The most prone to proteolysis was CH10, which was degraded right after serum addition. It was indicated by the appearance of a broad peak at the chromatogram. The MS analysis showed that the peak consisted of an overlapping peaks corresponding to constituent peptides, HNP[Cys5] and LFamB-C. This was a result of disulfide bridge reduction by glutathione present in serum. During the incubation time, the peaks assigned to constituent peptides were also vanishing. They were prone to proteolysis also when investigated separately. 58%, 51% and 0% of HNP[Cys5] was



detected after 1, 2 and 24 hours of incubation. On the contrary, 82% of LFamB-C remained intact after 1 h incubation, but it was completely degraded after 2 hours. Stability of CH10 was also evaluated with bovine β-trypsin, under conditions where disulfide bond is not reduced. Unfortunately, similarly to the experiment with serum, CH10 was rapidly proteolyzed. As shown by HPLC and MS analysis, the conjugate was not present in the reaction mixture after one hour of incubation with trypsin (see Fig. S2 in supporting information). These results significantly differ from our previous stability studies of trypsin inhibitor SFTI-1 and its analogues.17,18 We have shown that intramolecular disulfide bonds in SFTI-1 and its analogues were not reduced in serum, and their presence in molecules significantly increased proteolytic resistance of these compounds.18

Conformational analysis CD spectra of chimera peptides and their constituent peptides recorded in water and trifluoroethanol (TFE) are shown in Fig. 1. The CD spectra of all three HNP peptides (HNPK, HNP[Cys5] and HNP[Abu5]-K) (Fig. 1A) recorded in water revealed a minima around 205 nm and a negative ellipticity in the range of 206 – 219 nm which suggest that they adopt a bent structure.19 This can be associated with the presence of 2-aminobenzoic acid (2Abz) residue in the sequence of HNP, intentionally introduced by Lundy et al.12 as bent-forming mimetics. The shapes of all three CD spectra are very similar and observed differences may indicate different content (the highest for peptide with Abu and the lowest with Cys in position 5) of ordered structure. The CD spectra of LFampB and LFampB-C recorded in water (Fig. 1B) exhibit minima bellow 200 nm which are characteristic for random coil conformation. Significantly, more negative ellipticity was developed for the peptide extended with Cys at the C-terminus. It should be noted that this effect is not associated with dimerization or oxidation of the LFampB-C, as one could expect, having in mind that a single thiol group in studied peptide was presented. MS analysis of LFampB-C and HNP[Cys5] showed that under experimental conditions both peptides remained unmodified. CD spectra of CH8 and CH9 (Fig. 1C), similarly to LFampB, displayed minima bellow 200 nm representing rather unordered structure. For CH8, the negative ellipticity is significantly lower than that observed for the constituent LFampB. In case of CH9, the CD spectrum is almost identical with that obtained for LFampB. It is worth to mention that in both chimeras, the impact of the second constituent peptide (HNP-K or HNP[Abu5]-K, respectively) on their secondary structures seems to be rather limited. In case of CH10, a minimum was shifted to longer wavelengths and a strong negative ellipticity in the range of 200 – 218 nm is observed


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which can be assigned to the presence of bent structure. The content of the ordered structure seems to be higher than for the constituent peptide (HNP[Cys5]). These results clearly showed that the type of linker (isopeptide bond in CH8 and CH9 or disulfide bridge in CH10) used for connection of peptide chains influence not only chemical nature (non-degradable versus redox-sensitive linker) but also secondary structure of the synthesized chimera peptides. CD spectra of discussed peptides recorded in TFE are shown in Figs. 1D – 1F. This solvent is widely used to mimic biological membranes.20 Shapes of CD spectra of all three HNP peptides (Fig. 1D) display maxima at 191 – 194 nm and minima at 205 nm. As compared to those obtained in water, their intensities are lower, and maxima and minima are shifted to longer wavelengths, suggesting higher content of ordered structure. Generally, TFE did not induce significant changes in the secondary structures of these peptides. The CD spectrum of

Fig. 1. CD spectra of chimera peptides CH8, CH9, CH10 and their constituent peptides recorded in H2O (A –C) and trifluoroethanol (D – F). The concentration of all studied peptides was 18 µM.

LFampB recorded in TFE (Fig. 1E) exhibited maximum at around 200 nm, minimum at 203 nm and negative ellipticity between 204 and 240 nm which can be associated with the presence of a helical structure of the studied peptide. The extension of LFampB with Cys caused significant change in the CD spectrum recorded in TFE. A strong negative ellipticity in the range of 200 – 240 nm, with the minimum at 204 nm and a shoulder at 222 nm was developed. The shape of this spectrum indicates the high content of a α-helical structure. The CD spectrum of CH8 recorded in TFE (Fig. 1F) is characterized by the minimum at 203 nm and broad negative ellipticity in the between 200 and 240 nm and resembled that obtained for constituent peptide LFampB. This means that under experimental conditions, CH8 adopted



helical structure preferentially. Substitution of Cys(Acm) by Abu in CH8 yielded chimera CH9 with reduced helical content in its secondary structure in the solvent studied. This was indicated by the shift of a minimum to shorter wavelengths and less negative ellipticity in the range of 200 – 240 nm. Distinctly different CD spectrum, was recorded for CH10. It was distinguished by the maximum at 192 nm, a strong negative ellipticity in the range of 200 – 240 nm with a minimum shifted to 205 nm. The shape of the spectrum is very similar to the one recorded for constituent peptide LFampB-C, suggesting that in TFE chimera peptide CH10 adopts well defined helical structure. Although, it should be noted that the helical content in CH10 is lower than in constituent peptide LFampB. This probably reflects the contribution of the bent structure of constituent peptide, HNP[Cys5] in the secondary structure of CH10. The results presented above correspond well with the conformational analysis of a hybrid peptide LFchimera composed of LFcinB and LFampinB published by Bolscher et al.13 CD spectra of these peptides were recorded in HEPES buffer, pH 7.4 and in the presence of liposomes formed by dimyristoylphosphatidylglycerol (DMPG). CD spectra of the chimera and constituent peptides recorded in aqueous solution were interpreted as representing a random coil conformation, whereas in DMPG liposomes helical structures for both LFampinB and LFchimera, and a β-turn for LFcin were revealed. These results suggest that LFcin, when transferred from aqueous solution to the environment that mimics biological membrane, adopt ordered structure (presumably β-turn type IV). Our results showed that the three-dimensional structure of HNP peptides is less susceptible for the solvent, and they adopt bent conformation (more likely β-turn type I or III19) also in aqueous solution. A helical structure (N-terminal αhelix and a flexible cationic C-terminus) of LFampB in the environment that mimics biological membranes (SDS micelles) was confirmed by Haney et al.10 by more accurate method (1H-NMR in conjunction with theoretical calculations). Under conditions that mimic biological membranes, LFchimera, and described here CH8 and CH9, all containing isopeptide bond as a linker, adopt similar, helical conformation. Significantly higher helical content was displayed by CH10. In this compound, two peptide chains were connected via disulfide bridge. The obtained results clearly show the correlation between helical character of chimera peptides and their antibacterial activity. Antifungal activity The three chimera conjugates and their constituent peptides were tested for growth inhibitory activity against three fungal stains (C. albicans ATCC 10231, C. albicans SC 5314, ACS Paragon Plus Environment

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C. glabrata DSM 11226). None of the studied compounds exhibited antifungal activity at concentrations up to 100 µg/mL. These results are partly inconsistent with the literature data reported by other authors.9,10 Specifically, LFampB was previously found active against C. albicans ATCC 10231, with LC50 (the concentration that caused 50% reduction in viable counts) obtained after 1 h incubation equal to 4.3 µg/mL.9,10 In our study we determined the antifungal effect after 24 h incubation, according to the CLSI recommendations. On the other hand, in case of the HNP-K oligopeptide, Lundy et al. reported lack of activity against C. albicans.12 Antibacterial activity Antibacterial in vitro activity of compounds studied was determined against four bacterial strains, two Gram-positive (S. aureus 25923, S. epidermidis 12228) and two Gramnegative (E.coli 25922, P. aeruginosa 27853). Results of this assay are summarized in Table 2 and 3. Table 2. MIC values determined for selected bacterial strains. MIC [µg/mL] Strains Compound

S. aureus

S. epidermidis

E. coli

P. aeruginosa

MIC

MIC50

MIC

MIC50

MIC

MIC50

MIC

MIC50

LFampB

>> 250

>> 250

>> 250

>> 250

>> 250

>> 250

>> 250

>> 250

LFampB-C

>> 250

250

62

62

>> 250

>> 250

>> 250

>> 250

HNP-K

>> 250

>> 250

>> 250

125

250

250

>> 250

>> 250

HNP[Cys5]

>> 250

>> 250

>> 250

250

>> 250

>> 250

>> 250

>> 250

HNP[Abu5]-K

>> 250

31

>> 250

125

250

125

>> 250

>> 250

CH8 HNP-K-LFampB

>> 250

62

> 250

62

250

61

31

31

CH9 HNP[Abu5]-K-LFampB

>> 250

62

31

16

125

61

31

31

CH10 HNP[Cys5]-LFampB-C

>250

62

31

16

>> 250

250

>> 250

250

HNP-K+LFampB

>> 250

>> 250

> 250

250

>> 250

>> 250

>> 250

>> 250

HNP[Abu5]-K+LFampB

>> 250

250

> 250

250

>> 250

250

>> 250

>> 250

HNP[Cys5]+LFampB-C

>> 250

>> 250

>> 250

125

>> 250

>> 250

>> 250

>> 250

Ciprofloxacin (CIP)

0.02

0.01

0.63

0.31

0.005

< 0.005

0.08

0.08



Lewofloxacin (LVX)

0.02

0.01

0.63

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0.08

0.01

0.01

0.31

0.31

Table 3. MIC values determined for selected bacterial strains [µM]. MIC [µM] Compound

MW

Strain S. aureus

S. epidermidis

E. coli

P. aeruginosa

MIC

MIC50

MIC

MIC50

MIC

MIC50

MIC

MIC50

LFampB

2047.4

122.11

122.11

122.11

122.11

122.11

122.11

122.11

122.11

LFampB-C

2150.6

116.25

116.25

28.83

28.83

116.25

116.25

116.25

116.25

HNP-K

2081.4

120.11

120.11

120.11

60.06

120.11

120.11

120.11

120.11

1881.4

132.88

132.88

132.88

132.88

132.88

132.88

132.88

132.88

HNP[Abu ]- K

1991.8

125.51

15.56

125.51

62.76

125.51

62.76

125.51

125.51

CH8

4110.8

60.82

15.08

60.82

15.08

60.82

14.84

7.54

7.54

CH9

4021.2

62.17

15.42

7.71

3.98

31.09

15.17

7.71

7.71

CH10

4030

62.03

15.38

7.69

3.97

62.03

62.03

62.03

62.03

Ciprofloxacin (CIP)

331.3

0.06

0.03

1.90

0.94

0.02

0.02

0.24

0.24

Levofloxacin (LVX)

361.7

0.06

0.03

1.74

0.22

0.03

0.03

0.86

0.86

5

HNP[Cys ] 5

LFampB in our hands did not exhibit antimicrobial activity at concentrations up to 250 µg/ mL, whereas its LFampB-C analogue, containing the Cys residue at the C-terminus, inhibited growth of Gram-positive bacteria. Its activity was relatively weak against S. aureus but significantly higher against S. epidermidis. It should be stressed that the literature data indicated antimicrobial activity of LFampB against selected, both Gram-positive and Gramnegative bacteria.9,10 All three HNP-derived peptides exhibited moderate antimicrobial activity against three bacterial strains but were inactive against P. aeruginosa. The most pronounced activity was determined for HNP[Abu5]-K with MIC50 values 31 and 125 µg/mL against S. aureus and E.coli, respectively, but the concentration required for overall growth reduction was significantly higher. A reference peptide extended with Lys at its C-terminus (HNP-K) was less active and its analogue with free thiol group (HNP[Cys5]) exhibited only poor inhibitory activity against S. epidermidis. All three chimera peptides displayed significantly higher antimicrobial activity than the constituent peptides. CH8 and CH9 exhibited the most potent activity against all experimental strains, being the most effective against S. epidermidis and P. aeruginosa. Activity of CH10 against Gram positive bacteria was similar to that of CH8 and CH9 but this compound was practically inactive against Gram-negative strains. These results clearly show that the type of a linker used to connect AMPs determines the antibacterial activity of chimera peptides. Presence of the isopeptide ACS Paragon Plus Environment

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bond in chimeras CH8 and CH9 resulted in their remarkable antimicrobial activity against both Gram-positive and Gram-negative bacteria, whereas connection of the two peptide chains by a disulfide bridge afforded CH10, specifically active against Gram-positive bacteria. The fact that the equimolar mixtures of constituent peptides were practically inactive, clearly indicates that antibacterial activity of chimera peptides results from the presence of a covalent linkage between the constituent peptides. Cytotoxicity To assess the cytotoxic effect of CH9 on mammalian cells, we evaluated IC50 values (using MTT assay) for four human cell lines; non-cancer cell line of embryonic kidney (HEK 293) and three cancer cell lines: non-small cell lung cancer (A549), bone osteosarcoma (U-2 OS), and acute myeloblastic leukemia (HL-60). The response was measured either after 120hour (HL60) or 72-hour (A549 and U-2 OS) incubation. Figure 2 shows dose-response viability curves for A549 and U-2 OS. U-2 OS cell line was almost two times less sensitive to CH9 than A549. In case of HL60 cell line an additional comparison to well-established antifungal (FLC) and antibacterial (CIP and LVX) drugs was performed [manuscript in preparation] (Table 4). CH9 proved to be significantly less toxic than CIP, with IC50 value 1.6 times higher than the latter. At the same time IC50 values for FLU and LVX were around 3.5 and 1.8 times higher than for CH9, respectively. Sensitivity of HL60 cell line to CH9 was similar to A549 and nearly two times higher than for U-2 OS. Nevertheless, the concentration leading to 50% cell proliferation inhibition was high (tens of micromolar range) and comparable to antimicrobial drugs widely used in clinic. Therefore, it can be stated that the CH9 peptide induce a relatively low cytotoxic effect. Cell line A549

Cell line U-2 OS

120

120

IC50=25.70 ± 1.24 µΜ

80 60 40 20

IC50= 47.00 ± 1.07 µΜ

100

Survival [%]

100

Survival [%]

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


80 60 40 20

0

0

1

10

100

1000

1

Concentration [µΜ µΜ] µΜ

10

100

1000

Concentration [µ µΜ ]

Fig. 2. Dose-response viability curves for A549 and U-2 OS cell lines. Cells were seeded at 96-well plates at 1.5 × 103 and exposed to the action of CH9 for 72 h. Results obtained with MTT assay.



Page 18 of 32

Table 4. 120-hour response of HL60 cell line to CH9 compound compared to three wellestablished antimicrobial drugs.

IC50 ±SEM Compound µM

µg/ mL

CH9

23.03

±

4.74

92.6

±

19.9

CIP

14.30

±

2.45

4.7

±

0.8

LVX

41.39

±

7.80

15.0

±

2.8

FLC

80.50

±

10.92

24.7

±

3.3

Cytotoxic effect of CH9 as well as CH8 toward human non-cancer cell line HEK 293 was negligible as IC50 value was in both cases higher than the highest tested concentration (> 250 µM). Additionally, we evaluated CH8 and CH10 cytotoxicity toward two HL-60 sublines: primitive and standard. Primitive HL-60 was obtained by passaging cells at low cell densities.21 This fraction includes mainly leukemic stem cells (LSC) and progenitor cells (cells in early stage of differentiation: multipotent progenitor-like, common myeloid progenitor-like, oligopotent progenitor-like). Mutations of hematopoietic stem cell (HSC) may result in LSCs formation and the acquisition of self-renewal ability which is considered as AML (Acute Myeloid Leukemia) initiation. Standard HL-60 comprises cells at later stages of development and maturation, and senescent cells which do not possess self-renewal ability. Thus primitive cells are considered as more resistant than standard.21 IC50 value of CH8 was around 5 times higher for primitive cells than for standard (Fig. 3). Similarly in the case of CH10 IC50 value was nearly three times higher than for standard cells (IC50=100.8 µM and 37.98 µM respectively). Clinical and experimental data show that leukopoietic stages remain homologous to their corresponding developmental stages of healthy blood cells. Therefore our results strongly suggest that studied chimeras will remain weakly toxic for developing blood cells. In our studies we also observed that cytotoxic effect of tested chimeras is much higher toward all three cancer cell lines than toward healthy embryonic kidney cells. IC50 values for CH9 toward HL-60, A549 and U2-S were 23.03, 25.70 and 47.00 µM respectively, while for HEK293 that was > 250 µM. Table 5 shows selectivity indexes for CH9 alone and in


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comparison to commonly used antimicrobial drugs. Selective toxicity of lactoferricin and its derivatives to cancer cells was reported by several researchers.22–24 This could be explained by subtle differences between the cell membranes of normal and cancer cells. The most pronounced difference is much more negatively charged cell membrane in cancer cells.25 The relatively high levels of negatively charged molecules are likely to be more attractive to the cationic antimicrobial peptides, such as lactoferricin B or its variants. Therefore, these peptides bind to cancer cell membranes easily, leading to their death.26 CH8 120 100

Survival [%]

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


RI =

IC50 for primitive cell culture = 5.0 IC50 for standard cell culture

80

RI

60 40 20 0 1

10

100

Primitive IC50=53.16 µΜ

Primitive

Standard IC50=10.63 µΜ

Standard

1000

Concentration [µΜ µΜ] µΜ

Fig. 3. Comparison of cytotoxicity effect of CH8 on primitive and standard cell cultures. Table 5. Selectivity indexes for CH9: IC50/MIC of compounds for S. epidermidis (µM).

IC50 MICCH9 SICH9 MICCIP SICIP MICLVX SILVX

HEK 293 > 250

A549 25.7

U-2 OS 47.0

HL-60 23.0

6.1

3.0

24.7

12.1

27.6

13.5

7.7 > 32

3.3 1.9

131.6

13.5 1.7

147.1

15.1

ROS generation Two cell lines were selected for ROS (reactive oxygen species) generation analysis; HEK 293 the most resistant and HL-60 the most sensitive to the tested compounds. Concentrations were established experimentally (the highest concentration not causing lethal effect). Generation of ROS was measured with the use of CM-H2DCFDA molecular probe which becomes fluorescent after oxidation by free radicals within the cell (“ROS level” axis in Fig. 4, log scale). 7-AAD probe was used to distinguish dead or apoptotic cells (high red fluorescence on “7AAD axis” in Fig. 4, log scale) from living cells (low red fluorescence).



All three chimeras tested exhibit relatively low ROS generation. Nevertheless, a significant number of ROS (> 25%) was still detected after 3 h of incubation of HL-60 cells with CH8. Strikingly, similar situation was not observed in HEK293 cell line. Results indicate that tested chimeras may be potent antimicrobial agents generating relatively low amounts of reactive oxygen species which are responsible for worsening the pro-inflammatory response induced by bacterial membrane components. On the other hand Lupetti reports27 that the candidacidal activity of hLF(1-11) may result from the production of ROS as well as reduction of internal thiols, such as glutathione and thioredoxin, which protect cells from damage by ROS. Lupetti et al.27 recorded that addition of N-acetyl-L-cysteine (NAC), which is a precursor of glutathione and an ROS scavenger, significantly decreased the hLF(1-11) induced candidacidal activity. Thus small amount of ROS generated during exposure to chimeras is in agreement with previously stated antimicrobial activity.

Fig. 4. ROS generation. HEK 293 CH8 and CH9 chimeras were added in the concentration equal to 150 µM. HL-60 cell line cells were treated with the concentrations 25 µM for CH8 and 50 µM for CH9 and CH10. Positive control was tested with 250 µM of H2O2. A panel twoparameter dot plot (7-AAD – apoptosis level, CM-H2DCFDA oxidisation -ROS level both in log scale). B ROS levels for both cell lines in comparison to controls. Cellular uptake of CH10 and its constituents


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Using carboxyfluorescein labeled chimera (CH10-CF) and its constituents we checked weather compounds undergo cellular uptake. Microscopic observations showed that CH10-CF as well as its constituent LFampB-C penetrate well into S. epidermidis cells. In contrast, the same effect was not observed for HNP[Cys5]-CF (Fig. 5). These results clearly show that the most probable reason for the lack of activity of HNP (MIC value >> 250 µg/mL) is the poor cell penetration.

Fig. 5. Fluorescence microscope images of CH10-CF and its constituents (CF-LFampB-C and HNP[Cys5]- CF) uptake by S. epidermidis. Cells were grown to the mid-log phase and exposed to the action of the compounds at concentration 200 µM for 30 min. Conclusions Three chimera

peptides composed of two antimicrobial peptides,

bovine

lactoferrampin and synthetic, truncated analogues of human neutrophil peptide 1 were designed and synthesized. Under experimental conditions, they demonstrated significantly higher antimicrobial activity than constituent peptides. Chimeras containing the isopeptide bond as a linker (CH8 and CH9) were active against both Gram-positive and Gram-negative bacteria, whereas the one with a disulfide bridge (CH10) was specifically active against Gram-positive bacteria. The observed specificity correlated well with a significantly higher content of the helical structure determined in an environment that mimics a biological



membrane in case of the chimera CH10 in compare to remaining two chimeras. The antimicrobial activity of the equimolar mixtures of studied peptides was lower than that of the individual peptides and chimeras. This observation indicates a lack of the synergistic effect of constituent peptides and importance of the covalent linkage between them for the enhancement of antibacterial activity. In this study we also reported that antimicrobial activity depends strongly on cell penetration. Microscopic observations showed that HNP[Cys5] with the lowest activities neither penetrates the cell nor interact with cell membrane. Cytotoxicity of tested chimera peptides against human cells is low and rather limited to cancer cells which possess low negative charge (in contrast to no-cancer which are neutral or slightly positive28). Indeed, selective toxicity of chimera peptides against pathogenic bacteria is also associated with negative charge of their cell surface. We also showed that generation of ROS in human cells is limited what can be perceived as an advantage from the point of view of eventual toxicity to the host. Nevertheless, small amount of ROS generated may be advantageous in combating pathogenic bacteria. Type of the linker has a significant impact on the proteolytic stability of studied compounds which is an important issue to consider especially in case of peptides. Taking into consideration both antimicrobial activity and selective cytotoxicity not affecting normal cells and also other presented results, we claim that such chimera peptides are very promising lead structures for the development of antimicrobial drugs. Materials and methods Peptide synthesis All peptides were generated using standard 9-fluorenylmethoxycarbonyl (Fmoc) chemistry at 50 µmole scale using Prelude Peptide Synthesizer (Protein Technology, Inc., USA). Each peptide was synthesized on a Tenta Gel S RAM resin (loading 0.24 mmol/g, Rapp Polymere, Germany) to produce C-terminal amide groups after cleavage. The peptide chain was elongated in the consecutive cycles of deprotection and coupling. Deprotection was performed with 20% piperidine in N,N-dimethylformamide (DMF), and peptide chains elongation

was

performed

using

N,N,N,N`-tetramethyl-O-(benzotriazol-

1yl)uroniumtetrafluoroborate(TBTU)/ 1-hydroxybenzotriazole (HOBt)/N-methylmorpholine (NMM) and three-fold molar excess of each N-α-Fmoc protected amino acid (GL Biochem, Shanghai, China), including the fluorophore 5(6)-carboxyfluorescein (Novabiochem, Merck Germany), Fmoc-2-Abz (Bachem, UK) and Fmoc-Lys(Mtt) (Novabiochem, Merck, Germany). In case of HNP containing 2-aminobenzoic acid (2-Abz), due to the weak nucleophilic nature of its amino group, the automatic synthesis was stopped after the coupling ACS Paragon Plus Environment

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of 2-Abz and followed by the attachment of the next protected amino acid Fmoc-Gln(Trt) manually in the oil bath at 37 °C. The isopeptide bonds in peptidic hybrids (CH8 and CH9) were formed between εamino group of Lys attached to the C-terminal amino acid residue of analogue of HNP-1, and α-carboxyl group of C-terminal amino acid residue of LFampB. Briefly, the synthesis was started by coupling of Fmoc-Lys(Mtt) to TentaGel S RAM resin, followed by the assembly of the full (including with N-α-Boc-protected Arg) peptide chain of HNP-1 analogue. Next, 4methyltrityl (Mtt), the orthogonal protection of the Lys ε-amino group, was removed with 2% TFA in dichloromethane (10 min at room temperature, repeated nine times) followed by the synthesis of LFampB amino acid sequence. In this way the chimera peptide comprises of two peptide chains coupled by the isopeptide bond. After completing the synthesis, peptides were cleaved from the resin and the protecting

groups

were

removed

in

one-step

procedure

using

a

mixture

of

TFA:phenol:triisopropylsilane:H2O (88:5:2:5, v/v/v/v). The crude peptides were purified by HPLC on Beckman Gold System (Beckman, USA) equipped with RP Supelco Discovery BIO, Wide Pore C8, column (10×250 mm, Sigma Aldrich) or by PLC 2050 Gilson HPLC with Gilson Glider Prep. Software (Gilson, France), equipped with Grace Vydac C18 (218TP) HPLC column (22×250 mm, 10 µm, 300 Å, Resolution Systems). The solvent systems were 0.1% TFA (A) and 80% acetonitrile in A (B). Different linear gradients were applied (flow rate 5.6 mLmin-1, monitored at 226 nm). The purities of the synthesized peptides were checked with HPLC Pro Star system (Varian, Australia) equipped with Kinetex 5 µm XBC18 100Å column (4.6×150 mm, Phenomenex®). The solvent system was as described above. Linear gradient from 10 to 90% B for 40 min, flow rate 1 mLmin-1, monitored at 226 nm was used. All described compounds showed purities of at least 95%. In order to confirm the correctness of molecular masses of the synthesized peptides, mass spectrometry analysis was carried out by MALDI MS (Biflex III MALDI-TOF spectrometer, Bruker Daltonics) with an α-cyano-4-hydroxycinnamic acid (CCA) and/or 2,5-dihydroxybenzoic acid (DHB) matrix. Intermolecular disulfide bridge formation Disulfide bridge which linked (HNP[Cys5] with LFampB-C was formed as described previously.29 In brief, HNP[Cys5] (0.01 mmol) in aqueous acetic acid (0.1 M, 4 mL) and 2,2’dithiodipyridine (0.02 mmol) in isopropanol (4 mL) were mixed and stirred for 1 h at ambient temperature (Fig. 6, upper panel line B). The peptide derivative containing activated thiol function was purified by RP-HPLC and lyophilized. Equimolar mixtures of dithiopyridyl-



peptide HNP[Cys5] and the LFampB-C, 0.02 mmol each, were dissolved in sodium acetate (0.1 M, pH 5, 6 mL) and stirred for 1 hour at room temperature (Fig. 6, upper panel line D). The resulting chimera (CH10) was purified by RP-HPLC and lyophilized (Fig. 6, upper panel line E).

Fig. 6. Intermolecular disulfide bridge formation in CH10. The reaction progress was monitored by HPLC and MS. Modification of LFampB-C by 2,2’dithiodipyridine at 0 min (A), and 60 min (B) and (F), reaction of activated LFampB-C and HNP[Cys5] after 60 min incubation (D) and (G), purified CH10 (E). Synthesis of labeled peptides 5(6)-carboxyfluorescein (Novabiochem, Merck Germany) was attached to the HNP peptide (HNP[Cys5]- CF) through the ɛ-amino group of Lys located at its C-terminus or to the α-amino group of lactoferrampin (CF-LFampB-C). After deprotection (removal of MTT with 2% TFA in dichloromethane) of the ɛ-amino group of Lys17 or α-amino group of the second peptide, a mixture of 5(6)-carboxyfluorescein, HATU, HOAt and DIPEA (molar ratio 1:1:1:2) in DMF was added. The reaction was carried out at room temperature overnight in dark. The procedure was repeated until the chloranil test gave a negative result. The peptides were removed from the solid support, with side-chain protecting groups and the crude peptides were purified as described above. To obtain the labeled CH10-CF, LFampB-C was linked with labeled HNP[Cys5]- CF by intermolecular disulfide bridge as described before. Peptide stability study in human serum


Page 24 of 32

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Studies of peptide stability in human serum were performed as described in.17 Human serum from male plasma was prepared for the assay as referred previously.30 The initial concentration of the peptide solutions was 3.2 mM. Each peptide solution was then diluted 10 times with human serum and incubated at 37 ºC. Incubation mixture was analyzed after 0, 1, 2, and 24 hours. At each time point, samples of 150 µL were taken and mixed with 150 µL of acetonitrile to separate the human serum proteins. Formed precipitate was removed by centrifugation for 5 minutes at 13,000 RPM. The obtained supernatants were analyzed by RPHPLC and MS after storing on ice for 1 h, applying conditions described above.17 In order to determine peptide stability at each time point, peptide peak area from recorded chromatogram was compared with the peptide peak area obtained just after mixing peptide and plasma, and expressed as its percentage. Each experiment was repeated twice. Circular dichroism For CD measurements, peptide solutions were prepared by weight from lyophilized material. The peptide concentrations were in the range of 18 – 36 µM. Since no significant difference in the shapes of the CD spectra were observed, the results showed in Fig. 1 were obtained for peptide concentration of 18 µM. CD spectra were recorded at room temperature on Jasco J-815 spectropolarimeter operated in the 190 – 260 nm range, automated and equipped with a software Jasco Spectra Manager, Quartz cells of 1 mm were used. The results were plotted as the mean residue ellipticity [Θ]r [degree×cm2×dmol-1]. Trifluoroethanol (TFE) was of spectroscopic quality. Microorganisms strains and growth conditions In order to determine the biological features of obtained compounds following strains were used: C. albicans ATCC 10231, C. albicans SC 5314, C. glabrata DSM 11226, E.coli ATCC 25922, P. aeruginosa ATCC 27853, S. aureus ATCC 25923, S. epidermidis ATCC 12228. Before every experiment all of the yeast strains were maintained on YPD agar plates (1% (w/v) yeast extract, 2% (w/v) peptone, 2% (w/v) glucose, 2% (w/v) agar) in 30 ºC and bacterial strains on LA (Luria-Bertani agar; 1% (w/v) tryptone, 1% (w/v) NaCl, 0.5% (w/v) yeast extract, 2% agar) plates in 37 ºC for 16 – 24 h. Assay of antifungal activity The in vitro growth inhibitory activity of antifungals was quantified by determination of MIC values by the serial two-fold dilution method, using the 96-well microtiter plates, in



two media: buffer RPMI-1640 (Roswell Park Memorial Institute 1640 Medium, SigmaAldrich) and YNB-AS (Yeast Nitrogen Base with ammonium sulfate, without amino acids, Sigma-Aldrich). Conditions of the RPMI-1640-based assay were the same as outlined in the CLSI recommendations (M27-A3 document, Clinical Laboratory Standards Institute 2008). MIC was established visually as the lowest concentration that prevents the growth of microorganisms. MIC50 was defined as the lowest drug concentration that gave at least 50% reduction of cell density in comparison with the drug-free control (MIC50). The 96-well microtiter plates were also used for determination of in vitro growth inhibitory activity in YNB-AS medium. Serial two-fold dilutions of tested compounds were prepared in 0.1 mL aliquots in individual wells. Candida cells from the overnight cultures in YPD liquid medium were washed with sterile water and suspended in YNB-AS to cell density OD660 = 0.1 (approximately 106 CFU mL-1) and then diluted 50 times with the same medium. Individual wells of the microtiter plates were inoculated with 0.1 mL aliquots of cell suspensions in YNB-AS, the inoculated plates were incubated at 37 °C for 24 h and then turbidity was measured with a microplate reader at 660 nm. The MIC and MIC50 values were defined analogously as shown above for the RPMI-1640-based assay. Assay of antibacterial activity Evaluation of antibacterial activity of the tested compounds was performed using a serial two-fold dilution method in 96-well microtiter plates method recommended by CLSI, described in M07-A10 document. Two-fold serial dilutions of the tested compounds were prepared in 0.1 mL aliquots in individual wells in MHB II (Mueller Hinton broth, cationadjusted, Sigma- Aldrich) medium. Plates were inoculated with 0.1 mL aliquots of bacterial cell suspensions in MHB II, resulting in the final cell density ∼ 4 × 105 CFU mL-1. Plates were incubated at 37 °C for 24 h. The MIC and MIC50 values were defined analogously as shown above for the assay of antifungal activity. Cellular uptake of CH10 and its constituents Uptake of fluorescently labeled peptides by S. epidermidis was followed by fluorescence microscopy. Bacterial cells were grown overnight at 37 ºC in LB medium with shaking (140 RPM). The overnight culture was diluted with fresh LB medium to achieve OD600 = 0.1 (~108 CFU mL-1) and the cell suspension was grown for ∼ 4 h to the mid-log phase (OD600~ 0.3). The fluorescently labelled peptides were added to cell suspension to the final concentration of 200 µM and incubation was continued for 30 min (37 ºC, 140 RPM).


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Cells were then harvested, washed three times with PBS solution and immobilized on poly-Llysine coated glass slides. The preparations thus obtained were observed using an Olympus IX83 fluorescence microscope equipped with a 60 × oil immersion lens under green light excitation. Determination of cytotoxicity Cell culture media, antibiotics (penicillin and streptomycin) and FBS (Fetal Bovine Serum, Ref 35-015-CV) were purchased from Corning. Stock solutions of CIP, LVX and FLC (Sigma-Aldrich) were freshly prepared by dissolving compounds in sterile water acidified with hydrochloride acid (CIP, LVX) or in DMSO (FLC). All cell lines were purchased from ATCC (American Type Culture Collection). Drug sensitivity parameters were determined for four mammalian cell lines: HL-60 (human promyelocytic leukemia cells), A549 (human lung carcinoma), U-2 OS (bone osteosarcoma) and HEK 293 (human embryonic kidney). Multiwell (96-well) plates were seeded at 5 × 103 HL-60 cells/well or 1.5 × 103 A549 cells/well in RPMI-1640 supplemented with 10% FBS or 1.5 × 103 U-2 OS cells/well in RPMI-1640 supplemented with 10% FBS or 103 HEK 293. Media were supplemented with 2 mM L-glutamine and antibiotics (62.6 µg/mL) and streptomycin (40.0 µg/mL). Three adherent cell lines A549, U-2 OS and HEK 293 were allowed to attach overnight in 100 µL aliquots. Compounds tested were dissolved in medium and added to wells in 100 µL aliquots of 2 × concentrated solutions ranging from 2500 (FLC) or 250 µM (CH8, CH9, CH10 and remaining antibiotics) of two-fold final concentrations. To control wells of adherent lines, DMSO was added at a final concentration of 1%. HL-60 and HEK 293 cells were incubated with studied compounds for 120 h while two remaining cell lines for 72 h at 37 °C and 95%/5% CO2 or 10% CO2 (HEK 293) atmosphere. Subsequently, 20 µL aliquots of MTT solution in PBS (4 mg/mL) were added to each well and plates were further incubated for 2-3 h at 37 °C. Absorbance was measured after solubilization of formazan crystals in 200 µL DMSO, using a multiwell plate reader at λ= 540 nm. Cell proliferation was determined compared to nontreated cells (% control). All experiments were performed three times independently, each in triplicate. ROS generation HEK 293 cell line was seeded at 15 000 cells/plate (35 mm Petri dishes) for 24 h in DMEM medium (Dulbecco's Modified Eagle Medium, Sigma-Aldrich). CH8 and CH9 chimeras were added in the concentration equal to 150 µM. HL-60 cell line cells were plated



at the density of 25 000 cells/mL in RPMI-1640 medium and treated with the concentrations 25 µM for CH8 and 50 µM for CH9 and CH10. Positive controls were treated with 250 µM H2O2. Negative controls were left without addition of any compound. Following 0.5, 2.5, 5.5 and 24 h incubation cells were exposed to the action of 1 µM CM-H2DCFDA (5-(and-6)chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate, acetyl ester *mixed isomer) (Thermo Fischer Scientific, Waltham, USA) molecular probe for additional 0.5 h. HEK 293 cells were detached with 0.05% trypsin solution in PBS and HBSS (Hank’s Balanced Salt Solution) and suspended in fresh DMEM medium. All the samples were treated with 7-AAD (7aminoactinomycin D) in final concentration 0.8 µg/mL. Analysis were repeated three times and performed with Guava easyCyte flow cytometer (Merc, Burlington, USA). Acknowledgement This work was supported by the National Science Centre (NCN) under grant No UMO2016/21/B/ST5/00101. Supporting Information 1. Conjugates and peptides stabilities in human serum. 2. HPLC chromatograms of mixture obtained during incubation of CH10 with bovine βtrypsin. Abbreviations Abu, L-α-aminobutyric acid; Abz, aminobenzoic acid; Acm, acetaminoethyl; AMPs, antimicrobial peptides; CH, chimera; CIP, ciprofloxacin; CD, circular dichroism; CF, 5(6)carboxyfluorescein; FLC, fluconazole; HNP, human neutrophil peptide; IC50, 50% inhibitory concentration; LF, lactoferrin; LFampB, bovine lactoferrampin; LVX, levofloxacin; MIC, minimum inhibitory concentration; PBS, phosphate-buffered saline References (1)

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Antimicrobial Activity of Chimera Peptides Composed of Human

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