Development of New Antimicrobial Agents from Cationic PG

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Development of New Antimicrobial Agents from Cationic PG-surfactants containing oligo-Lys peptides Ryosuke Kimura, Masahide Shibata, Shuhei Koeda, Atsushi Miyagawa, Hatsuo Yamamura, and Toshihisa Mizuno Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.8b00693 • Publication Date (Web): 24 Oct 2018 Downloaded from http://pubs.acs.org on October 26, 2018

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Bioconjugate Chemistry

Development of New Antimicrobial Agents from Cationic PGsurfactants containing oligo-Lys peptide Ryosuke Kimura, Shibata Masahide, Shuhei Koeda, Atsushi Miyagawa, Hatsuo Yamamura, and Toshihisa Mizuno* Graduate School of Engineering, Nagoya Institute of Technology, Gokiso-cho Showaku, Nagoya, Aichi 466-8555, Japan *Corresponding author: Tel. & Fax: +81-52-735-5237; Email: [email protected]

1 ACS Paragon Plus Environment

Bioconjugate Chemistry 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

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ABSTRACT Peptide gemini-surfactant (PG-surfactant), a kind of lipopeptide, is composed of a short linker peptide (X) between two alkyl-chain-modified Cys residues, and peripheral peptides at the N-terminal (Y) and the C-terminal (Z) sides, respectively, of the alkylated Cys residues. In this study, we developed and examined a series of PG-surfactants containing two C12 saturated alkanes and oligo-Lys, arranged at the X-, Y-, or Z-positions. To arrange oligo-Lys at the Y- or Z-positions, a repeat sequence of -Asp-Lys-Asp-Lyswas used at the X-position. All of the PG-surfactants exhibited high antimicrobial activity against both gram-positive and negative bacteria. In addition to high antimicrobial activity, a low hemolysis activity is prerequisite for efficient intravenous administration. Among the synthesized PG-surfactants, those having -(Lys)3- at the Y- or Z-positions, i.e. K3-DKDKC12 and DKDKC12K3, showed reasonably low hemolytic activities. This combination of high antimicrobial activity along with low hemolytic activity is an essential and unique property, and has not been previously reported for the synthesized lipopeptides. Further, using scanning electron microscope (SEM) and N-phenyl-1naphthylamine (NPN) uptake assay we showed that the antimicrobial activity of these PG-surfactants may be attributed to membrane disruptive mechanisms. Although the PGsurfactants with low hemolytic activity could interact and localize onto red blood cell surfaces, and cause slight expansion of cell morphologies, no subsequent penetration occurred. In summary, we describe here the successful development of PG-surfactants having high antibacterial and low hemolytic activity, thus providing a significant molecular platform to develop novel antimicrobial agents.


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INTRODUCTION Since bacterial infection is implicated in serious diseases such as Hansen's disease,1 tuberculosis,2 tetanus,3 infective endocarditis,4 organ abscesses,5 and acute pharyngitis,6 the antimicrobial reagents that can combat various pathological bacteria are important and indispensable for maintenance of health. However, because bacteria inherently have an ability to develop resistance against various antimicrobial reagents via genetic mutation(s) or fusing exogenous genes, there is considerable demand for the development of new antimicrobial reagents.7 Several naturally occurring lipopeptides with antimicrobial activity have been identified in fungi and microbes.8 Since the Food and Drug Administration’s recent approval of daptomycin as a new antibiotic, effective against vancomycin-resistant Staphylococcus aureus,9 there has been renewed interest in lipopeptide-based antibiotics. Studies have examined chemical derivatives of natural lipopeptides for improvement in antimicrobial activity and specificity. However, because of the inherent complexity in molecular structures, brought about by non-ribosomal translation and post-translational modifications, chemical derivatizations of these natural lipopeptides requires multi-step organic synthesis; therefore, significant improvements have not yet been achieved.10 Although application of molecular evolution techniques to the parent lipopeptide molecules using modification enzymes has been suggested,11 there is a need for novel simple molecular framework to design antimicrobial lipopeptides. Meanwhile, we recently studied the evolution of several functional molecules from peptide-gemini surfactants (PG-surfactants). These PG-surfactants (Figure 1) are a kind of lipopeptide having a gemini molecular structure, which consists of two alkyl-chainmodified cysteine residues, a short linker peptide (-X-) between them, and periphery peptides at the N- and C-terminal sides (Y-, and –Z, respectively).12 In our previous study, by choosing the suitable peptide sequences for -X-, Y-, and -Z, we succeeded in designing bilayer-forming amphiphiles,12,13 that could act as solubilization surfactants,14,15 and extraction surfactants16 for membrane proteins. Interestingly, in spite of a simple basic molecular framework, we found that there is a clear relationship between the peptide sequences at X-, Y-, and Z-positions and the resulting properties and functions of the PGsurfactant. Such versatile nature of PG-surfactants prompted us to design a novel class of antimicrobial lipopeptides from the PG-surfactant molecular scaffold. Generally, it is well known that cationic surfactants can bind cell surfaces well and through membrane disruption cause cytotoxicity. For example, the C16 alkyl chains modified with Lys-Lys, 3 ACS Paragon Plus Environment


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Lys-Gly-Lys, and Lys-Lys-Lys-Lys were reported to show antimicrobial activities.17 However, for practical use in intravenous therapy, in addition to high antimicrobial activity, a low hemolytic activity is also necessary. Therefore, in this study we aimed to develop a series of cationic antimicrobial PG-surfactants, containing oligo-Lys or oligoArg at -X-, Y-, or –Z positions, and having low hemolysis activity. Using microscopic techniques and NPN uptake assay, we further discuss about the working mechanism of antimicrobial PG-surfactants for bacterial and red blood cells.


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RESULTS AND DISCUSSION Design of antimicrobial PG-surfactants Due to the anionic nature of the cell walls and cell membranes of bacteria (originating from anionic phospholipids such as phosphatidylglycerols and cardiolipin, lipoteichoic acid polymers, and sugar chains in membrane proteins), cationic surfactants are known to exhibit effective antimicrobial activity via membrane disruptive mechanisms.18 Based on this general principle, we designed a series of cationic PGsurfactants and examined them for antimicrobial activity. The peptide sequences arranged at the X-, Y-, and Z-positions of the PG-surfactants are summarized in Table 1. According to our previous study, the suitable number of amino acid residues at the X-position in PGsurfactants is 3 or 4.12 Therefore, we first prepared cationic PG-surfactants having (Lys)3- and -(Lys)4- at the X-position, called K3C12 and K4C12, respectively. To compare the impacts of introducing cationic residues at the other two positions, we prepared DKDKC12K3 and K3-DKDKC12 that has Ac-(Lys)3- or –(Lys)3-NH2 at the Y- and Zpositions, respectively. The -Asp-Lys-Asp-Lys- sequence, utilized at the X-position in DKDKC12K3 and K3-DKDKC12, is a betaine sequence having a neutral net charge, and therefore it might be effective to clarify the impact of cationic charges at the Y- and Zpositions on antimicrobial activity. As reference molecule for these cationic PGsurfactants, molecule having anionic charges at the Y-position (DKCKC12D5) was also examined. To clarify the necessity for two C12 chains in one molecule, those having one C12 chain and one C1 chain (DKDKC12C1K3, and DKDKC1C12K3) were also examined. To further understand the relationship between molecular structure and antimicrobial activity, we also prepared and examined the following molecules: that containing Ac(Arg)3- instead of Ac-(Lys)3- (DKDKC12R3), those containing oligo-Lys with different number of Lys residues, Ac-(Lys)n- (n = 1, 2, 4, 5) (DKDKC12Kn, n = 1, 2, 4, 5), and molecule having oligo-Lys at both N- and C-termini (K3-DKDKC12K3).



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Characterization of PG-surfactants Firstly, we characterized the fundamental micelle formation properties of the PGsurfactants by measuring the critical aggregation concentration (CAC)19 and hydrodynamic diameter in a buffer. The obtained data are summarized in Figure 2 and Table 2. Similar to DKDKC12K, DKDKC12D, and NPDGC12KK that we reported previously,14,16 most PG-surfactants, having two C12 chains and the -Asp-Lys-Asp-Lyssequence at the X-position, showed CACs of 7-15 µM. In contrast, the PG-surfactants with one C12 and one C1 chains (DKDKC12C1K3, and DKDKC1C12K3) showed CAC values more than 1 mM. This suggests that the gemini-type molecular structure is necessary to exhibit low CAC. Interestingly, the CAC values of PG-surfactants, K3C12 and K4C12, having oligo-Lys at the linker peptide (-X-) showed significant larger CAC values at 5040 and 11500 µM, respectively.14 Through dynamic light scattering (DLS) measurements, we found that at concentrations above their CACs (500 µM or 2 mM), all PG-surfactants formed 6-8 nm molecular assemblies (Figure 2). Consistent with previous reports, 14,16 these PG-surfactants formed only micelle-like assemblies under the wide concentration range used, and did not form any molecular aggregates such as fibers, sheets, or lamellar aggregates.

NH

HN

O

O S

Y Cys

S X

Cys Z

Figure 1. Chemical structure of PG-surfactants with C12 alkyl chains 6 ACS Paragon Plus Environment

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Table 1. Summary of the peptide sequences introduced at -X-, Y-, and Z- positions and the type of alkyl chains of PG-surfactants PG-surfactants

Peptide sequence

Peptide sequence

Peptide sequence

at Y-

at -X-

at -Z

Alkyl chain at

Alkyl chain at

the N-terminal

the C-terminal

side of Cys

side of Cys

Total charge at neutral pH

K3C12

Ac-

-(Lys)3-

-NH2

C12

C12

+3

K4C12

Ac-

-(Lys)4-

-NH2

C12

C12

+4

DKDKC12K

Ac-Lys-

-Asp-Lys-Asp-Lys-

-NH2

C12

C12

+1

DKDKC12K2

Ac-(Lys)2-

-Asp-Lys-Asp-Lys-

-NH2

C12

C12

+2

DKDKC12K3

Ac-(Lys)3-

-Asp-Lys-Asp-Lys-

-NH2

C12

C12

+3

DKDKC12K4

Ac-(Lys)4-

-Asp-Lys-Asp-Lys-

-NH2

C12

C12

+4

DKDKC12K5

Ac-(Lys)5-

-Asp-Lys-Asp-Lys-

-NH2

C12

C12

+5

DKDKC12D5

Ac-(Asp)5-

-Asp-Lys-Asp-Lys-

-NH2

C12

C12

-5

DKDKC12C1K3

Ac-(Lys)3-

-Asp-Lys-Asp-Lys-

-NH2

C12

C1

+3

DKDKC1C12K3

Ac-(Lys)3-

-Asp-Lys-Asp-Lys-

-NH2

C1

C12

+3

DKDKC12R3

Ac-(Arg)3-

-Asp-Lys-Asp-Lys-

-NH2

C12

C12

+3

K3-DKDKC12

Ac-

-Asp-Lys-Asp-Lys-

-(Lys)3-NH2

C12

C12

+3

-Asp-Lys-Asp-Lys-

-(Lys)3-NH2

C12

C12

+6

K3-DKDKC12-K3 Ac-(Lys)3-



Figure 2. DLS profiles of

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DKDKC12K3 (500 µM), DKDKC12R3 (500 µM), K3C12 (2

µM), DKDKC12D5 (500 µM), DKDKC12C1K3 (2 mM), and DKDKC1C12K3 (2 mM) in a buffer (20 mM phosphate buffer (pH 7) was used except for K3C12. 20 mM Acetate buffer (pH 5) was used only for K3C12).


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Table 2. Critical aggregation concentration (CAC, µM and µg/mL) and hydrodynamic diameters (d, nm) of PG-surfactants in 20 mM phosphate buffer (pH 7) CAC (µM)

CAC (µg/mL)

d (nm)a

K3C12

5040b,c

5550 b,c

7b

K4C12

11500b,c

14100 b,c

7b

DKDKC12K

8.3c

11

7

DKDKC12K2

14

20

6

DKDKC12K3

13

21

7

DKDKC12K4

11

19

7

DKDKC12K5

11

20

6

DKDKC12D5

36

64

6

DKDKC12C1K3

1430

1970

6d

DKDKC1C12K3

1330

1830

6d

DKDKC12R3

10

17

6

K3-DKDKC12

11

17

6

K3-DKDKC12K3

37

73

5

PG-surfactant

a

These data were observed for 500 µM of PG-surfactants except for K3C12, K4C12,

DKDKC12C1K3, and DKDKC1C12K3 in 20 mM phosphate buffer (pH 7). b

These data were observed for 2 mM of K3C12 and K4C12 in 20 mM acetate buffer (pH

5). c

These data are cited from our previous study.14

d

These data were observed for 2 mM of DKDKC12C1K3 and DKDKC1C12K3 in 20 mM

phosphate buffer (pH 7).



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Evaluation of antimicrobial and hemolytic activities of the PG-surfactants Antimicrobial activities of the cationic PG-surfactants were evaluated from the minimum inhibitory concentrations (MICs)20 for the representative gram-positive and negative bacteria, S. aureus and B. subtilis, and E. coli and S. typhimurium, respectively. The results are summarized in Figures 3, Figure S1, Figure S2 and Table 3. As we expected, the cationic PG-surfactants consisting of oligo-Lys or oligo-Arg at -X-, Y-, or -Z and two C12 chains, exhibited antimicrobial activities against both gram-positive and -negative bacteria. Such a wide antimicrobial spectrum displayed by the cationic PGsurfactants is consistent with the typical property of surfactant-based antimicrobial reagents.21 Interestingly, those lacking one C12 chain, such as DKDKC12C1K3 and DKDKC1C12K3, did not show any antimicrobial activity (> 512 µg/mL) for the grampositive or -negative bacteria, suggesting that the gemini-type molecular structure having two C12 chains is indispensable for induction of high antimicrobial activity. By comparing MIC and CAC values of the PG-surfactants, we could guess the active species contributing to the antimicrobial activity. The CAC values of PG-surfactants, K3C12 and K4C12, having oligo-Lys at the linker peptide (-X-), have larger CACs at 5040 and 11500 µM, respectively but it exhibited high antimicrobial activity (MIC for gram-positive and -negative bacteria < 32 µg/mL (29 µM for K3C12 and 26 µM for K4C12)). It implied that monomeric specie of PG-surfactants contribute to antimicrobial activity. DKDKC12Kn, (n = 2-5), DKDKC12R3, K3-DKDKC12, and K3-DKDKC12K3 have lower CACs by 8-15 µM than K3C12 and K4C12, but still these are larger than their MICs. It meant that all antimicrobial PG-surfactants exhibited antimicrobial activity by their monomeric species. Less antimicrobial activity was observed for those lacking one C12 chain (DKDKC12C1K3 and DKDKC1C12K3). This seems to be contrasting to a previous study that examined lipopeptides consisting of cationic peptides and one C16 chain, where significant antimicrobial activity was attributed to nanoaggregates of cationic lipopeptides due to the single C16 chain.17 However, in our study, the DLS measurements of DKDKC12C1K3 and DKDKC1C12K3 indicated that these PG-surfactants showed less micelle formation ability and did not form nanoaggregates over the concentration range of their CMCs. This difference could be due to the difference in the alkyl chain lengths, and weaker hydrophobic interaction among C12 chains than C16 chains might cause differences in antimicrobial activity. In terms of the position of oligo-Lys, those introduced at -X- position (K3C12 and K4C12), Y-position (DKDKC12K3), -Z-position 10 ACS Paragon Plus Environment

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(K3-DKDKC12), and both Y- and Z-positions (K3-DKDKC12K3), all resulted in similar antimicrobial activities. These data support that a combination of cationic oligo-Lys and C12 gemini-structure is necessary and sufficient for high antimicrobial activity. By comparing DKDKC12Kn (n = 1-5) having a different number of Lys residues at the Y-position, we examined relationship between the number of Lys residues and antimicrobial activity. In case of molecules with 3–5 Lys residues (DKDKC12K3, DKDKC12K4, and DKDKC12K5), a higher antimicrobial activity (MIC < 16 µg/mL) was exhibited for both gram-positive and -negative bacteria. In contrast, decreasing the number of Lys residues to 1 or 2, resulted in a decrease in the antimicrobial activity, especially for gram-negative bacteria. These data suggest that a minimum number of 3 Lys residues are required for effective antimicrobial activity. Further, gram-negative bacteria have outer and inner membranes around the outer edge of the cells, which is stronger than those of gram-positive bacteria. Thus, reduced antimicrobial activity against gram-negative bacteria is consistent with a membrane-disruption mechanism underlying the antibacterial activity.22 For practical application of the antimicrobial PG-surfactants as prescribed drugs, it is essential to have the molecules display minimum side effects. To ensure minimum damage to red blood cells is one of the essential requirements prior to in vivo test as an antimicrobial agent for intravenous administration. Generally, cationic surfactants such as CTAB disrupt cell membranes not only of bacterial cells but also of red blood cells.21 Previously, attempts were made to accomplish selective disruption of cell membranes through discrimination between bacterial and red blood cells; however, it proved to be quite challenging with low success rate.23 In this study, we evaluated the hemolytic activity (HC50) of the cationic PG-surfactants using rabbit red blood cells as an alternative to human red blood cells (Table 3).24 Surprisingly, unlike general cationic surfactants, the cationic PG-surfactants having oligo-Lys at the Y-position, such as DKDKC12K3, DKDKC12K4, and DKDKC12K5, showed significantly low hemolytic activity. These PG-surfactants, therefore, may be selective for bacterial cells. Although some amphiphilic a-helical peptides are known to exhibit high antimicrobial activity and low hemolysis,25 we believe that this is the first example of non-natural lipopeptides showing both high antimicrobial along with low hemolytic activity. Unlike those having oligo-Lys at Y-position, the K3C12 and K4C12, that have oligo-Lys at the -X- position showed high hemolysis. We further examined the 11 ACS Paragon Plus Environment


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HC50 values of K3-DKDKC12, having (Lys)3 at the Z-position and K3-DKDKC12K3, having two (Lys)3 at both the Y- and Z-positions. We observed that these PG-surfactants showed no hemolysis activity (> 600 µM). Therefore, introducing oligo-Lys at Y- and/or Z-position seems to be important to suppress damages to red blood cells. Furthermore, in order to clarify relationship between choice of cationic amino acids and hemolysis, we also observed HC50 of DKDKC12R3 having oligo-Arg at the Y-position instead of oligoLys. The DKDKC12R3 molecule was found to exhibit high hemolytic activity, suggesting that presence of oligo-Lys is important to achieve selective bacterial cell damage. The reference

compounds,

those

lacking

one C12

chain

(DKDKC12C1K3

and

DKDKC1C12K3) and having oligo-Asp at the Y-position (DKDKC12D5), which displayed no antimicrobial activities, exhibited no hemolytic activity. These data suggest that lower hydrophobic and/or electrostatic interactions of PG-surfactants with cell membrane decrease hemolysis as well as antimicrobial activities.


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(a)

(b)

Figure 3. Comparison of the minimum inhibitory concentrations (MICs) (µg/mL) displayed by the PG-surfactants for E. coli, S. typhimurium, B. subtilis, and S. aureus (The upper (a) is the range of MIC 0-500 µg/mL and the lower (b) is the range of MIC 0140 µg/mL).


Bioconjugate Chemistry 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

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Table 3. Minimum inhibitory concentration (MIC) for gram-positive and negative bacteria and hemolytic activity (HC50) of PG-surfactants PG-surfactants E. coli

Minimum Inhibitory Concentration

Hemolytic

Hemolytic

MIC (µg/mL)

activity

activity

B. subtilis

HC50 (µM)

HC50 (µg/mL)

S. typhimurium S. aureus

K3C12

32

8

8

8

4

4

K4C12

4

8

4

4

7

9

DKDKC12K

512

> 512

32

128

24

32

DKDKC12K2

32

64

8

16

>600

>875

DKDKC12K3

8

16

4

4

>600

>952

DKDKC12K4

4

8

4

4

>600

>1030

DKDKC12K5

8

8

4

4

>600

>1110

DKDKC12D5

> 512

> 512

> 512

> 512

>600

>1070

DKDKC12C1K3

> 512

> 512

> 512

> 512

>600

>825

DKDKC1C12K3

> 512

> 512

> 512

> 512

>600

>825

DKDKC12R3

8

8

8

8

18

30

K3-DKDKC12

8

16

8

4

>600

>952

K3-DKDKC12-K3

8

16

4

---a

>600

>1180

Polymyxin B

1

1

64

8

---a

--- a

a

not observed


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Impacts of the PG-surfactants on cell morphologies of bacterial and red blood cells Most cationic PG-surfactants examined in this study showed high antimicrobial activity. Furthermore, the cationic oligo-Lys-based PG-surfactants such as DKDKC12K3, DKDKC12K4, DKDKC12K5, K3-DKDKC12, and K3-DKDKC12K3 could specifically damage bacterial cells and not red blood cells. In order to further understand the underlying mechanism of the cationic PG-surfactants on bacterial cells, we directly observed bacterial and red blood cells, before and after treatment with the antimicrobial PG-surfactants, using a scanning electron microscope (SEM) and laser scanning confocal microscope (LSCM). Firstly, we observed morphological changes in E. coli cells before and after treatment with the series of PG-surfactants. Farkas et al. have reported that plant antimicrobial peptides induced formation of blebs and/or debris on the surfaces of E. coli cells.26 The sample preparation of E. coli cells and the treatments were performed according to a previous report,25 and the obtained data are summarized in Figure 4. Unlike the untreated E. coli cells (Figure 4(A)), those treated with the PG-surfactants DKDKC12K5, DKDKC12K3, K3-DKDKC12, DKDKC12R3, K3C12, K4C12 and K3DKDKC12K3 showed clear emergence of debris on cell surfaces (Figure 4 (B), (D), (E), (G), (H), (I), and (L)). Among the different PG-surfactants, there were no differences observed in the degree of debris formation. Since the debris corresponded to leaked cell contents through damaged bacterial cell membranes, we postulated that the antimicrobial PG-surfactants could form pores not only in the outer membranes but also within the inner membranes of E. coli cells. This inference was reasonably supported from the results that the PG-surfactants with no

antimicrobial activities,

such

as DKDKC12D5,

DKDKC12C1K3, and DKDKC1C12K3, did not show any debris, similar to untreated cells (Figure 4 (C), (J), and (K)). Next, in order to elucidate why the cationic oligo-Lys-based PG-surfactants, having oligo-Lys at Y- and/or Z-position could achieve selective damage to bacterial cells, we observed red blood cells before and after treatment with the cationic PG-surfactants. As a representative of PG-surfactant showing high antimicrobial activity and low hemolysis, we chose DKDKC12K5. As a negative control, we chose DKDKC12R3, which displayed high antimicrobial and high hemolytic activity. In order to visualize the PG-surfactants by LSCM, both PG-surfactants were fluorescently labeled with dansyl or FITC groups at



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the N-terminal side. The obtained LSCM and SEM images are shown in Figures 5 and 6, respectively. Since red blood cells are non-nucleated, they are disc-shaped and slightly concave on both cell faces as shown in Figure 5 (a) and Figure 6 (a).27 Interaction with various chemicals is known to change this cell morphology to disc-shape with protrusions, expanded round-shape, or a center-hollowed doughnut-shape.27-29 Although treatment of the cells with DKDKC12K5 altered the morphology of red blood cells to an expanded round-shape (Figure 5 (b) and Figure 6 (b)), the cells remained intact. In contrast, treatment with DKDKC12R3 expanded the cells and caused them to burst (Figure 5 (c)). These data are consistent with differences in the measured hemolytic activities (Table 3). LSCM observations showed that DKDKC12R3 molecules were distributed within and on the surfaces of burst cells (Figure 5(c)). This implies that DKDKC12R3 molecules first penetrated inside the cells via disruption of membrane integrity and then induced the red blood cells to burst (Figure 5(c)). The DKDKC12K5 molecules were selectively localized only on the cell surfaces of red blood cells, with no penetration observed (Figure 5(b)). Thus, these data suggest that the PG-surfactants that achieved high antimicrobial activity and low hemolysis (such as DKDKC12K5) can interact with the red blood cell surfaces, but the cells were not lysed due to lower disruptive effect on cell membranes. Morphological alterations in red blood cells due to chemicals are known to recover once the concentration of the chemical decreases in the immersing buffer.27 In case of DKDKC12K5, similar recovery of cell morphology after washing in buffer was observed (Figure 6(c)). Such low and temporary side effects after treatment with DKDKC12K5 may suggest that it has potential to use as an antimicrobial drug possible to prescribe intravenous administration of the molecule.


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Figure 4. SEM observation of E. coli cells before and after treatment of the series of PGsurfactants; (A) no treatment, (B) treated with DKDKC12K5, (C) treated with DKDKC12D5, (D) treated with DKDKC12K3, (E) treated with K3-DKDKC12, (F) treated with DKDKC12K, (G) treated with DKDKC12R3, (H) treated with K3C12, (I) treated with K4C12, (J) treated with DKDKC12C1K3, (K) treated with DKDKC1C12K3, and (L) treated with K3-DKDKC12K3.



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Figure 5. Confocal microscopic observation of red blood cells (a) before and after treatment with (b) FITC-modified DKDKC12K5 and (c) Dansyl-modified DKDKC12R3; the excitation wavelengths are 488 nm for (b) and 405 nm for (c).


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Figure 6. SEM observation of red blood cells (a) before and (b) after treatment with DKDKC12K5, and (c) after washing the DKDKC12K5-treated cells in 10 mM HEPESNaOH buffer (pH 7.4) containing 150 mM NaCl; acceleration voltage 5 kV.



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Impact of PG-surfactants on the outer membrane of bacterial cells As discussed above, PG-surfactants having oligo-Lys at the Y- and/or Z-positions, such as DKDKC12K3, DKDKC12K4, DKDKC12K5, K3-DKDKC12, and K3-DKDKC12K, showed high antimicrobial activity along with low hemolytic activity, which is one of the essential requirements prior to in vivo test as an antimicrobial agent for intravenous administration. Furthermore, our observations indicated that it functions by disrupting the bacterial membrane, with the degree of impact being different for bacterial cell and red blood cell membranes, causing disintegration of only the former. Using conventional fluorescence assay of NPN uptake, we further examined the antimicrobial mechanism of the PG-surfactants on E. coli cells (Figure 7). NPN is a hydrophobic fluorescent molecule, whose fluorescence intensity increases upon binding hydrophobic regions of damaged cell membranes.30 In case of antimicrobial agents with no membrane disruptive mechanism, no increase in NPN intensity was observed. In Figure 7, the black circles means the time course of NPN fluorescence intensity after addition of the PG-surfactants (100 µM) or polymyxin B (100 µM) to a suspension of E. coli cells in a buffer and the blue circles means the time course of NPN fluorescence intensity after addition of saline (negative control) to a suspension of E. coli cells. As anther control, time course of NPN fluorescence intensity after addition of the PG-surfactants (100 µM) or polymyxin B (100 µM) in a buffer was also observed (red circles). Addition of DKDKC12K3 resulted in a 6-fold increase in the fluorescence intensity, and it saturated rapidly (Figure 7 (a)), suggesting that membrane damage could occur within seconds. Similar observations were recorded for the other antimicrobial PG-surfactants, as shown in Figure 7 (b) and (c). Polymyxin B, known to exhibit antimicrobial activity, was used as a positive control in Figure 7 (g) and showed similar increase in NPN intensity. In contrast, the nonantimicrobial PG-surfactants such as DKDKC12C1K3 and DKDKC1C12K3 did not cause any increase in NPN fluorescence (Figure 7 (e) and (f)). Treatment with DKDKC12D5, one of the non-antimicrobial PG-surfactants, showed an increase in NPN intensity (Figure 7 (d)). However, this was caused by preferential inclusion in micelle-like assemblies of DKDKC12D5, since NPN could not penetrate in E. coli cell membranes that has no damage by DKDKC12D5. Together, these data support our hypothesis that the antibacterial PG-surfactants function by disrupting the bacterial membrane, likely disrupting both outer and inner cell membranes. 20 ACS Paragon Plus Environment

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Figure 7. Time course of NPN fluorescence intensity at 410 nm (excitation at 340 nm) after addition of the PG-surfactants (100 µM) or polymyxin B (100 µM) in the presence of E. coli cells (black), after addition of the PG-surfactants (100 µM) or polymyxin B (100 µM) in the absence of E. coli cells (red), and after addition of saline to E. coli cells (blue). All NPN fluorescence intensities were normalized by the fluorescence intensity before addition of each PG-surfactant or polymyxin B; (a) DKDKC12K3, (b) DKDKC12R3, (c) K3C12, (d) DKDKC12D5, (e) DKDKC12C1K3, (f) DKDKC1C12K3, and (g) Polymyxin B.



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CONCLUSION In this study, we developed a novel class of antimicrobial reagents from a series of cationic PG-surfactants containing two C12 chains and oligo-Lys at the X-, Y- or Zpositions. Since the substitution of one C12 chain with a C1 chain in the PG-surfactants containing oligo-Lys did not show any antimicrobial activity, both the gemini-type molecular structure and the presence of two C12 chains with cationic oligo-Lys is indispensable for high antimicrobial activity. Furthermore, PG-surfactants having oligoLys at Y- and/or Z-position such as DKDKC12K3, DKDKC12K4, DKDKC12K5, K5DKDKC12, and K3-DKDKC12K3, successfully displayed low hemolytic activities, in addition to their high antimicrobial properties, which implied that it has potential to apply to therapeutic drugs possible to prescribe through intravenous administration. To the best of our knowledge, this is the first and rare example of synthesized lipopeptides that fulfills both these requirements. Further, unlike the naturally occurring antimicrobial peptides, the PG-surfactants have a simple molecular structure, whose framework can easily be used to adapt to therapeutic applications and chemical derivatization. Additionally, we hope to use this molecular framework to achieve further development of PG-surfactants displaying increased antimicrobial activity, selectivity, lower hemolysis, and lower cytotoxicity.


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EXPERIMENTAL PROCEDURES Materials Rink-amide AM resin (200–400 mesh) was purchased from Merck Biosciences (Darmstadt, Germany). N-(9-fluorenylmethoxycarbonyl) (Fmoc)-protected-amino acids, 1-hydroxybenzotriazole (HOBT), 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU), Rink-amide AM resin (200–400 mesh), N,Ndiisopropylethylamine (DIEA), piperidine, trifluoroacetic acid (TFA), and Nmethylpyrrolidone were purchased from Watanabe Chemical Industries (Hiroshima, Japan). Unless otherwise stated, other chemicals and reagents were obtained commercially and used without further purification. Synthesis of PG-surfactants PG-surfactants were synthesized on a Rink-amide AM resin using commercially available Fmoc-protected amino acids, and our synthesized Fmoc-Cys(C12)-OH.12,31 K3C12, K4C12 and DKDKC12K were synthesized according to our previous study.14 For condensation onto the resin, standard condensation reagents (HOBT/HBTU/DIEA) were used. The N-terminus of PG-surfactants was end-capped with Ac2O. To synthesize the dansyl-modified DKDKC12R3, the N-terminus was end-capped with dansyl chloride. To synthesize the FITC-modified DKDKC12K5, the N-terminus was first condensed with Fmoc-GABA-OH. After deprotection of Fmoc group, anew N-terminus was end-capped with FITC. After cleavage of the synthesized PG-surfactants from the resin using TFA/H2O (95/5), the crude PG-surfactants were purified by reversed-phase highperformance liquid chromatography with a core-shell-type ODS column (Kinetex, Shimadzu, Japan). A linear-gradient of CH3CN and H2O, both including 0.1 vol% TFA, was utilized as eluent. The purity of each sample was checked by high-resolution ESITOF (electrospray ionization time-of-flight) mass spectroscopy. DKDKC12K2: HRMS (ESI-TOF, [M + H]+): calcd. for C68H128N15O15S2, 1458.9156; found, 1458.9160. DKDKC12K3: HRMS (ESI-TOF, [M + H]+): calcd. for C74H140N17O16S2, 1587.0105; found, 1587.0094.



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DKDKC12K4: HRMS (ESI-TOF, [M + H]+): calcd. for C80H152N19O17S2, 1715.1055; found, 1715.1044. DKDKC12K5: HRMS (ESI-TOF, [M + H]+): calcd. for C86H164N21O18S2, 1843.2005; found, 1843.2001. DKDKC12D5: HRMS (ESI-TOF, [M + H]+): calcd. for C76H129N16O28S2, 1777.8604; found, 1777.8590. DKDKC12R3: HRMS (ESI-TOF, [M + H]+): calcd. for C74H142N23O16S2, 1673.0446; found, 1673.0458. DKDKC12C1K3: HRMS (ESI-TOF, [M + H]+): calcd. for C61H115N16O15S2, 1375.8169; found, 1375.8170. DKDKC1C12K3: HRMS (ESI-TOF, [M + H]+): calcd. for C61H115N16O15S2, 1375.8169; found, 1375.8193. K3-DKDKC12K3: HRMS (ESI-TOF, [M + H]+): calcd. for C92H176N23O19S2, 1971.2954; found, 1971.2957. K3-DKDKC12: HRMS (ESI-TOF, [M + H]+): calcd. for C74H140N17O16S2, 1587.0105; found, 1587.0078.

Dynamic light scattering measurements of PG-surfactant assemblies The concentrations of each PG-surfactant in 20 mM phosphate buffer (pH 7.0) were set at 500 µM and the mean hydrodynamic diameters of PG-surfactant assemblies at 25°C were estimated using a Zetasizer Nano ZS (Malvern Instruments, Ltd., Malvern, UK). Determination of the critical aggregation concentration (CAC) Fluorescence spectral changes of 8-anilino-naphthalene-1-sulfonic acid (ANS) before and after incorporation into micelle-like assemblies of the PG-surfactants in 20 mM phosphate buffer (pH 7) were used to evaluate CACs.38 The CAC values were calculated from the double linear-fitting analysis for F475. Evaluation of antimicrobial activities against gram-positive and -negative bacteria Minimum inhibitory concentrations (MICs) were determined by broth microdilution method.20 In this study, we used E. coli (K12, W3110) and S. typhimurium (LT2) as representative gram-negative bacteria and B. subtilis (B. subtilis 168) and S. aureus (FDA


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209P) as representative gram-positive bacteria. The bacteria were cultured in a medium (polypeptone (1 g/100 mL) with 34 mM NaH2PO4, 64 mM K2HPO4, 20 mM (NH4)2SO4, 1 µM FeSO4, 1 µM ZnCl2, 0.3 mM MgCl2, and 10 µM CaCl2 for E. coli and S. typhimurium; Luria Broth (LB) medium (polypeptone (1 g), yeast extract (0.5 g), and NaCl (0.5 g) in 100 mL) for B. subtilis; lactose bouillon (3 g/100 mL) for S. aureus) at 37°C overnight, after which cells were collected by centrifugation and suspended in 10 mL of physiological saline (0.7 – 1.0 X 106 CFU/mL) to prepare a stock solution of the bacterium cells. The stock solutions of the PG-surfactants with different concentrations (0.04, 0.08, 0.16, 0.32, 0.64, 1.28, 2.56 mg/mL) in sterilized water were separately prepared. In order to determine MICs, 20 µL of the bacterial stock solution, 75 µL of Mueller-Hinton medium, and 5 µL of the different concentrations of the PG-surfactant stock solution were mixed in a 96-well plate and cultured at 37°C for 20 h. Based on bacterial proliferation and colony forming abilities, we determined MIC for the different PG-surfactants. Polymyxin B (Wako Pure Chemical Industries Ltd., Japan), known to have high antimicrobial activity, was used as a positive control. Evaluation of hemolysis activity (HC50) of PG-surfactants Hemolytic activities of the PG-surfactants were evaluated as described before.23 Rabbit blood (2 mL) was mixed with physiological saline (8 mL) and subjected to centrifugation (2200 rpm, 10 min, 4°C) to separate blood plasma and the eliminated hemoglobin in supernatant from the precipitant of red blood cells. After removing the supernatant, the precipitant of red blood cells was suspended in physiological saline (8 mL) and subjected to centrifugation (2200 rpm, 10 min, 4°C) to separate residual blood plasma and the eliminated hemoglobin. After this double treatment, the red blood cells were finally suspended in 10 mM HEPES-NaOH buffer (pH 7.4) containing 150 mM NaCl to prepare 10 % hematocrit solution. To evaluate the degree of hemolysis by each PG-surfactant, 50 µL of PG-surfactant solution in 10 mM HEPES-NaOH buffer (pH 7.4) containing 150 mM NaCl and defined PG-surfactant concentrations (10, 50, 100, 200, 300, and 600 µM), 50 µL of 10 % hematocrit solution, and 900 µL of 10 mM HEPES-NaOH buffer (pH 7.4) containing 150 mM NaCl were mixed in a 1.5 mL tube and incubated at 37°C for 30 min with 90 strokes/min. The eliminated hemoglobin due to hemolysis was determined from A540 values. Using 50 µL of 50 µM L-α-lysophosphatidylcholine from egg yolk (Wako



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Pure Chemical Industries Ltd., Japan), the A540 for 100 % hemolysis of 50 µL of 10 % hematocrit solution was determined. SEM observation of E. coli and bacterial cells25, 27 For SEM observation of E. coli cells, E. coli cells were first cultured in 2 mL LB medium at 37°C for 16 h. After collection of cells by centrifugation, the cells were washed thrice in PBS. The E. coli cells were finally suspended in 100 µM of each PG-surfactant in PBS and incubated at 4°C for 20 h. Post-incubation and removal of the PG-surfactant solution by centrifugation, the cells were treated with 2 % glutaraldehyde in PBS at 4°C for 20 h to perform the first immobilization. After washing in PBS for three times, the second immobilization was carried out by treatment of the cells with 1 wt% osmium tetroxide in PBS at 4°C for 30 min. Dehydration of E. coli cells was further performed by stepwise treatment of the immobilized cells with different concentrations of ethanol (30, 50, 70, 90, 95, 100 %) solutions in PBS. The cells were then pasted onto an observation plate and subjected to Pt vapor desorption. SEM observation was performed with an accelerated voltage of 5 or 10 kV. As control, we carried out SEM observation of E. coli cells without PG-surfactant treatment, (incubated with only PBS at 4°C for 20 h). For SEM observation of red blood cells, as mentioned above for hemolytic activity evaluation, a 10 % hematocrit solution was first prepared from rabbit blood. To treat the red blood cells with each PG-surfactant, 1.8 mL of the 10 % hematocrit solution was mixed with 0.2 mL of PG-surfactant (500 µM) in 10 mM HEPES-NaOH buffer (pH 7.4) containing 150 mM NaCl and incubated at 37°C for 30 min with 90 strokes/min. After centrifugation (2200 rpm, 10 min, 4°C), the supernatant was removed and the precipitated red blood cells were washed three times in 10 mM HEPES-NaOH buffer (pH 7.4) containing 150 mM NaCl. The precipitated red blood cells were treated with 2 % glutaraldehyde in 10 mM HEPES-NaOH buffer (pH 7.4) containing 150 mM NaCl at 4°C for 20 h to perform the first immobilization. After washing three times in PBS, the second immobilization was applied by treatment of cells with 1 wt% osmium tetroxide in PBS at 4°C for 30 min. Dehydration of red blood cells and further steps for SEM observation were performed as described for the E. coli cells above. As control, we carried out SEM observation of red blood cells incubated with only PBS 37°C for 30 min with 90 strokes/min.


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Confocal laser-scanning microscope observation of red blood cells after treatment with PG-surfactants A 50 µL aliquot of the prepared 10 % hematocrit solution from rabbit blood was mixed with 50 µL of FITC-modified DKDKC12K5 or dansyl-modified DKDKC12R3 (50 µM) in PBS and incubated at 37°C for 30 min with 90 strokes/min. After diluting tentimes by adding 10 mM HEPES-NaOH buffer (pH 7.4) containing 150 mM NaCl, this solution was moved to a glass bottom dish and fluorescence images were acquired using an LSM880 microscope (Zeiss, German). Samples were imaged using a 63 X objective lens. Excitation of the dansyl or FITC groups in the PG-surfactants was carried out using the 405 nm or 488 nm lasers, respectively. To detect emission fluorescence of dansyl or FITC, in the PG-surfactants, we used the band-pass filters of 451–661 nm and 493–634 nm, respectively. NPN uptake assay for E. coli cells with or without treatments with PG-surfactants The 100 µL of suspension of E. coli cells in 5 mM HEPES-NaOH buffer (pH 7.2) (4.5 X 108 CFU/mL) was dispensed in each well of 96 well plate and 10 µL of NPN in acetone (0.2 mM) was added, respectively. After addition of the series of PG-surfactants with the defined concentrations (final concentrations is 100 µM), alteration of NPN fluorescence intensity at 410 nm (excitation at 340 nm) was traced using a fluorescent plate reader (SH-9000Lab, Hitachi-Hitech, Japan). ACKNOWLEDGEMENTS This work was supported by JSPS KAKENHI (Grant Numbers 17K05932), and the Ogasawara Foundation for the Promotion of Science & Engineering.

Supporting Information Available: Figures S1-S2, plus HPLC and ESI-MS data of the series of PG-surfactants. This material is available free of charge via the Internet at http:// pubs.acs.org.



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Table of Contents Graphic


Development of New Antimicrobial Agents from Cationic PG

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