Amino Terminal Copper and Nickel Binding Motif Derivatives of

Nov 27, 2017 - Each peptide was able to bind DNA and RNA with micromolar affinity, but did not display nuclease activity in vivo. The ATCUN OV-3 deriv...
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Cite This: J. Med. Chem. 2017, 60, 10047−10055

Amino Terminal Copper and Nickel Binding Motif Derivatives of Ovispirin‑3 Display Increased Antimicrobial Activity via Lipid Oxidation Jessica L Alexander, Zhen Yu, and J. A Cowan* Evans Laboratory of Chemistry, The Ohio State University, 100 West 18th Avenue, Columbus, Ohio 43210, United States S Supporting Information *

ABSTRACT: Antimicrobial peptides are short peptides secreted by the innate immune system to protect the host from pathogens. We have investigated the influence of the amino terminal copper and nickel binding (ATCUN) motif on derivatives of ovispirin-3 (OV-3), an αhelical peptide from the cathelicidin family, demonstrating an increased antimicrobial activity toward a broad range of bacteria, relative to OV-3, with MICs as low as 1.3 ± 0.6 μM. Each peptide was able to bind DNA and RNA with micromolar affinity, but did not display nuclease activity in vivo. The ATCUN OV-3 derivatives also displayed an increased membrane leakage and lipid peroxidation relative to Cu-GGH and OV3 alone. These data suggest that the Cu-ATCUN derivatives inhibit bacteria by binding to the membrane, promoting oxidative damage of the lipids, which then disrupts the bilayer, resulting in cell death. This stands in contrast to the mode of action of OV-3 alone, which permeabilizes the membrane without lipid oxidation.

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INTRODUCTION Antimicrobial peptides (AMPs) are a large group of naturally occurring peptides found in all living organisms, of which hundreds have been classified.1−6 AMPs play a role in the innate immune response by defending the host against pathogens.1−6 They are able to target and inhibit the growth of many infectious agents, including Gram-positive and Gramnegative bacteria, yeast, and fungi, and are of interest as a new source of antimicrobial agents due to an increasing resistance to standard antibiotics.1−6 AMPs typically contain a large number of hydrophobic and positively charged residues arranged in an amphipathic design, and many exhibit an extended conformation or random coil structure in aqueous solution. Following interaction with membranes, they form well-defined secondary structure motifs, suggesting that they are able to bind to the bacterial membrane.7−10 Other studies have shown an effect on the fluorescence response from tryptophan-carrying peptides when incubated with lipids, further supporting the hypothesis that AMPs are able to target bacterial membranes.8 Several peptides are also able to induce pore formation in the membrane, including tritrpticin9 and temporins.11 In addition to targeting the plasma membrane, there is increasing evidence that AMPs are able to target intracellularly located biomolecules. Enzymatic assays have demonstrated that CP10A and indolicidin inhibit aminoglycoside phosphotransferases and acetyltranserases,12 while Sub5 and Bac2A are able to directly interact with ATP to inhibit ATP-dependent enzymes.13 Previous work has demonstrated that the presence of an amino terminal copper and nickel binding (ATCUN) motif in © 2017 American Chemical Society

AMPs can increase their biological activity toward pathogens. The ATCUN motif is a physiologically relevant sequence that is most commonly found in albumins.14 Coordination of copper or nickel requires histidine in the third position as well as two deprotonated amide backbone nitrogen atoms. The ATCUN motif coordinates both metals in a square-planar geometry and the copper atom undergoes a 2+/3+ redox cycle. Histatin-3 and histatin-5 are both members of the salivary histatin family and contain an ATCUN motif within their sequence. Analogues of histatin-5 were able to bind DNA and exhibit nuclease activity.15 ATCUN derivatives of anoplin led to lipid peroxidation via an increased ROS generation,16 while ATCUN derivatives of buffering are able to cleave DNA.17 The 29-residue sheep myeloid antimicrobial peptide (SMAP29) is an α-helical cathelicidin that is potent against a range of pathogens and acts by disrupting the cellular membrane.18 Ovispirin 3 (OV-3) is a truncated derivative of ovispirin (OV), a synthetic derivative of SMAP-29, and is also part of the cathelicidin family with a broad-range antimicrobial activity toward numerous pathogens.1,3 OV includes an amidated Cterminal unlike SMAP-29 and has an increased number of hydrophobic residues to increase the α-helical character of the peptide (Table 1). OV-3 contains the sequence from residues 3 to 16 of OV with the first residue as Ile instead of Leu and is also amidated (Table 1). Although the biological activity of OV-3 has not been characterized, it is thought to act in a similar manner as SMAP-29 since the overall α-helical character of the Received: August 4, 2017 Published: November 27, 2017 10047

DOI: 10.1021/acs.jmedchem.7b01117 J. Med. Chem. 2017, 60, 10047−10055

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Table 1. AMPs Used in This Study peptide

sequence

SMAP-29 OV OV-3 1 2 3 4

RGLRRLGRKIAHGVKKYGPTVLRIIRIAG KNLRRIIRKIIHIIKKYGPTILRIIRIIG-NH2 IRRIIRKIIHIIKK-NH2 GGHGIRRIIRKIIHIIKK-NH2 GGHGIRRIIRKIIHIIKKGGC-NH2 GGHIRRIIRKIIHIIKK-NH2 GGHIRRIIRKIIHIIKKGGC-NH2

Table 2. Summary of Secondary Structure Determined by CD for OV-3 Derivatives peptide OV-3 Cu-1 Cu-2 Cu-3 Cu-4

peptide is maintained. However, previous studies have demonstrated differences in MICs due to differences in the amino acid sequence of cathelicidins, so small changes can have a drastic effect on the ability of the peptide to target the cell.19,20 We have found that derivatives of OV-3 containing the ATCUN motif (GGH) (Table 1) display enhanced antimicrobial activity relative to OV-3 and by a distinct mode of action. These peptides contain the ATCUN motif (GGH) as well as the OV-3 sequence. These peptides were also able to bind and cleave plasmid DNA but did not result in any DNA cleavage in vivo. Furthermore, these peptides were able to bind the 16S Asite rRNA, but had no nuclease activity toward the RNA. Although Cu-GGH and OV-3 were unable to permeabilize the plasma membrane or promote oxidative membrane damage in Escherichia coli, Cu-ATCUN derivatives of OV-3 containing a C-terminal GGC sequence displayed high levels of membrane permeabilization and lipid peroxidation. ATCUN OV-3 derivatives lacking this C-terminal sequence were also able to oxidize lipids, but did not display enhanced antimicrobial activity due to a decrease of α-helical structure. These data suggest that Cu-ATCUN derivatives of OV-3 confer their increased antimicrobial activity via lipid peroxidation that results in disruption of the bacterial membrane.

phosphate

SDS micelles

% β-sheet

% α-helix

% β-sheet

% α-helix

8.51 8.95 5.16 8.83 3.50

19.08 9.44 24.56 12.60 29.18

2.62 9.22 0.14 2.31 0.07

37.69 13.78 55.58 35.17 63.71

the most pronounced secondary structure at 24.56% and 29.18% α-helix in phosphate buffer. In SDS micelles, these peptides all display minima at 210 and 222 nm, and their overall α-helical structure increases, with Cu-2 and Cu-4 again showing the most significant secondary structure. No major differences were observed for any of the ATCUN-derivatives following the addition of copper. Previous work has shown that SMAP-29 and ovispirin derivatives form an α-helix in hydrophobic environments while exhibiting no structure in phosphate buffer.21,22 Antimicrobial Activity. A microdilution assay was employed to determine the susceptibility of various bacterial strains toward the Cu-ATCUN OV-3 derivatives (Cu-AMPs). The bacterial strains were grown overnight, diluted to (1−7) × 105 CFU/mL, and then incubated with 2-fold serial dilutions of Cu-AMPs, with the highest concentration at 16 μM. Following overnight incubation, the MIC was recorded as the lowest amount of complex at which no visible growth occurred (Table 3). Cu-2 and Cu-4 exhibit an increased antimicrobial activity against E. coli 25922, Pseudomonas aeruginosa, methicillinresistant Staphlyococcus aureus (MRSA) 43300, Streptococcus pneumoniae, and Enterococcus faecium, relative the other ovispirin AMPs (Table 3). Conversely, Cu-1 and Cu-3 exhibited the lower MICs for Klebsiella pneumoniae and Acinetobacter baumannii, which are both Gram-negative organisms. This trend is likely a reflection of the ability of Cu-2 and Cu-4 to form dimers (Figure S12), which results in an increased secondary structure formation, relative to Cu-1 and Cu-3 (Figure 1), and most likely results in more effective targeting of microorganisms. Metallopeptide Cu-4 also had the lowest MIC toward K. pneumoniae (8.0 ± 0.0 μM). Aside from A. baumannii, Cu-4 had the lowest MIC for every organism tested, with the lowest MIC observed for E. coli 25922 (1.3 ± 0.6 μM). This discrepancy in the potency of the Cu-AMPs between Gramnegative and Gram-positive bacteria most likely reflects the difference in permeability of the Gram-negative organisms that results from their variable membrane composition; a wellrecognized issue with regard to discovering novel antibiotics.23−25 Many peptides bind to and disrupt the bacterial membrane as their mechanism of action. SMAP-29 is able to bind to lipopolysaccharides (LPS), and its MIC is correlated with its binding affinity.22 Furthermore, the N-terminal α-helical region of SMAP-29 is required for its antimicrobial activity.21 Cu-4 had the lowest MIC of all of the peptides for each microorganism studied except for A. baumannii; this peptide also displayed the most defined α-helical structure in SDS micelles, suggesting that enhanced structure plays a role in its biological activity. Cu-4 also has an increased α-helix character and enhanced antimicrobial activity compared to Cu-2, which

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RESULTS AND DISCUSSION Peptide Design. For all experiments, peptide X refers to the peptide in the Cu-free form, while Cu-X refers to the peptide with copper bound to the ATCUN motif. Peptide 2 was purchased from AAPPTec, while the other peptides were synthesized through solid-phase peptide synthesis using standard Fmoc chemistry. Following purification by RP HPLC (Figure S1), the purity of each peptide was verified by ESI mass spectrometry (Figure S2). Each metallopeptide contains the GGH metal binding domain, which facilitates chemistry, and the original sequence of OV-3, which binds to the therapeutic target and provides selectivity. The combination of the ATCUN motif with the OV-3 targeting domain has the potential for irreversible inactivation of biological targets and multiple turnover activity. Cu-1 and Cu-2 contain a glycine linker between the metal binding and targeting domains in order to investigate differences in chemistry due to distinct spacial arrangements of the metal binding and targeting domains. Metallopeptides Cu-2 and Cu-4 both contain a Cterminal GGC sequence for later conjugation of molecular motifs through the Cys thiol for applications such as drug delivery. Structural Characterization. Table 2 lists a summary of the secondary structure elements observed for each peptide by circular dichroism (CD) spectroscopy and evaluated by the use of the K2D3 program. Figure 1 shows that OV-3, Cu-1, and Cu-3 all have an α-helical character, but Cu-2 and Cu-4 display 10048

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Figure 1. CD spectra of OV-3 (a), peptide 1 (b), peptide 2 (c), peptide 3 (d), and peptide 4 (e) in phosphate buffer and SDS micelles.

Table 3. MICs for OV-3 Cu-AMPs against Various Bacterial Strainsa complex

E. coli 25922

P. aeruginosa

K. pneumoniae

A. baumannii

MRSA 43300

S. pneumoniae

E. faecium

CuCl2 Cu-GGH OV-3 Cu-1 Cu-2 Cu-3 Cu-4

>16.0 >16.0 >16.0 >16.0 3.0 >16.0 1.3

>16.0 >16.0 16.0 >16.0 12.0 >16.0 4.0

>16.0 >16.0 >16.0 16.0 >16.0 16.0 8.0

>16.0 >16.0 0.7 2.7 9.3 5.3 10.7

>16.0 >16.0 >16.0 16.0 4.0 16.0 1.5

>16.0 >16.0 16.0 16.0 8.0 8.0 4.0

>16.0 >16.0 16.0 16.0 8.0 8.0 3.3

OV-3 and Cu-AMPs were incubated with each microorganism overnight at 37 °C using a microdilution assay. The highest concentration tested was 16 μM, and the MIC reported is the lowest concentration that showed no visible bacterial growth.

a

⎛ ⎛ x ⎞ p⎞ y = (y2 + (y1 − y2 )/⎜⎜1 + ⎜ ⎟ ⎟⎟ ⎝ KD ⎠ ⎠ ⎝

contains an additional glycine linker between the ATCUN and OV-3 domains of the peptide. Therefore, it appears that a linker between the two domains leads to a decrease in structure and antimicrobial activity for these two peptides. Previous studies have demonstrated that OV-3 shows a broad range of antimicrobial activity,3 whereas our data demonstrates weak antimicrobial activity for OV-3 toward microbes, with the exception of A. baumannii. DNA Binding and Nuclease Activity. A gel shift assay was employed to determine the affinities of the metallopeptides for pUC19. Cu-AMPs were incubated in the presence of pUC19 for 1 h with up to a 6-fold excess of metallopeptide and then analyzed on a 1% agarose gel (Figures 2 and S4). A plot of the percent unbound pUC19 vs log[Cu-AMP] was fit to a logarithmic function (eq 1) to yield KD (Figures 2 and S5). In eq 1, y1 is the initial concentration of the unbound nucleic acid, y2 is the final concentration of unbound nucleic acid, p is the cooperativity of the transition, and KD is the binding affinity. OV-3 derivatives were found to bind pUC19 with micromolar affinity, with Cu-2 displaying the lowest KD value of 3.8 ± 1.1 μM, suggesting that these complexes may bind intracellular nucleic acid targets. There was no correlation between charge and binding affinity, or rate constants for cleavage and binding affinity.

(1)

The ability of Cu-AMPs to oxidatively damage DNA was also tested with pUC19 in the presence of ascorbate and hydrogen peroxide for up to 1 h. The reaction products were then analyzed on a 1% agarose gel (Figure S6), and the intensities of the bands were quantified and fit to eqs 2−4 to determine the rate constants for nicking and linearization (Table 4, Figures 3 and S7). Metallopeptides Cu-2 and Cu-4 showed high levels of plasmid cleavage, similar to the positive control containing CuCl2. These two peptides were also able to produce linearized DNA. [S] = [S]0 e−k1t

(2)

⎛ k 1 ⎞ −k t [N ] = [S]0 ⎜ ⎟(e 1 − e−k 2t ) + [N ]0 (1 − e−k 2t ) ⎝ k 2 − k1 ⎠ (3)

10049

[L] = [L]0 e−k 2obst

(4)

flin = n2e−n2

(5)

fsup = e[−(n1+ n2)]

(6) DOI: 10.1021/acs.jmedchem.7b01117 J. Med. Chem. 2017, 60, 10047−10055

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Figure 3. (a) Agarose gel from time-dependent reactions of pUC19 with Cu-4 and (b) plots for supercoiled (black), nicked (red), and linear (green) pUC19 over time. Solutions of 100 nM Cu-AMPs were incubated with 10 μM pUC19, 1 mM ascorbate, and 1 mM hydrogen peroxide in 20 mM HEPES and 100 mM NaCl, pH 7.4. Reactions were run for up to 1 h at room temperature and then separated by agarose gel electrophoresis.

Figure 2. (a) Agarose gel shift assay for Cu-1 with pUC19 and (b) a plot of % unbound pUC19 vs log[Cu-AMP] used to calculate the binding affinity. Cu-AMPs were incubated with 10 μM pUC19 up to a 6-fold excess and allowed to equilibrate for 1 h at room temperature before separation on an agarose gel. The relative intensities of the bands for the unbound pUC19 were used to determine the KD value for each peptide.

the other peptides had similar binding affinities in the micromolar range. The ability of the Cu-AMPs to oxidatively damage Fl-16S rRNA was also examined by incubating each complex with RNA in the presence of hydrogen peroxide and ascorbate for up to one hour. Reaction mixtures were then separated by agarose gel electrophoresis (Figures 5 and S10), and the intensities of the bands corresponding to unbound Fl-16S rRNA were used to determine the amount of RNA remaining at each time point. Plots of [16S rRNA] vs time were fit to an exponential decay function (eq 7) to calculate the rate constants for RNA nicking. Figure 5 shows the resulting gel and plot of [Fl-16S rRNA] vs time for Cu-2, and Table 4 summarizes the rate constants for nicking. Cu-2 was the only complex to show reactivity toward RNA, with a rate of 39.3 ± 14.3 min−1 × 103. This rate is much slower compared to the rate of duplex DNA cleavage for this complex.

To determine if the metallopeptides promoted cleavage of DNA via a conserved mechanism, a Freifelder−Trumbo analysis was conducted for each of the time-dependent reactions (eqs 5 and 6). The ratio of double-stranded breaks to single-stranded breaks (n2/n1) is related to the fractions of supercoiled and linearized DNA. If n2/n1 is greater than 0.01, then the metallopeptide is able cleave both strands via a consecutive mechanism rather than by random nicking of both strands to produce linearized DNA. With the exception of Cu3, all of the metallopeptides have n2/n1 values greater than 0.01. Metallopeptide Cu-4 displayed the highest n2/n1 ratio of 0.110 ± 0.060, which correlates with its high rate of DNA nicking and linearization, supporting a consecutive model of DNA cleavage. RNA Binding and Nuclease Activity. The ability of OV-3 and metallopeptides to bind 16S A-site rRNA was also examined. The conditions for the assay were similar to those used to determine the binding affinity for pUC19 (Figures 4, S8, and S9) and are summarized in Table 4. Cu-1 displayed the highest binding affinity with a KD value of 4.5 ± 1.2 μM, while

(−(x − x0)/ t ) y = y0 + ye i

(7)

Table 4. Summary of of Nucleic Acid Binding and Nuclease Activity for OV-3 Derivatives complex

KD for pUC19 (μM)

knicking for pUC19 (min−1 × 103)

CuCl2 Cu-GGH OV-3 Cu-1 Cu-2 Cu-3 Cu-4

>60 >60 8.4 ± 1.1 13.4 ± 1.1 3.8 ± 1.1 5.2 ± 1.0 5.9 ± 1.1

273.4 ± 47.6 86.1 ± 19.8 N/A 97.2 ± 6.7 231.5 ± 75.1 72.3 ± 4.6 245.7 ± 60.2

n2/n1 0.045 0.052 N/A 0.026 0.049 0.001 0.110

± 0.032 ± 0.044 ± ± ± ±

0.026 0.021 0.001 0.060 10050

% cells FITC positive

KD for Fl-16S rRNA (μM)

knicking for Fl-16S rRNA (min−1 × 103)

± ± ± ± ± ± ±

>60 >60 7.3 ± 1.2 4.5 ± 1.2 10.4 ± 1.3 10.5 ± 1.1 11.3 ± 1.1

no reaction no reaction N/A no reaction 39.3 ± 14.3 no reaction no reaction

44.1 33.7 3.7 7.5 4.7 3.8 6.0

16.6 10.3 1.6 1.1 5.6 1.3 4.6

DOI: 10.1021/acs.jmedchem.7b01117 J. Med. Chem. 2017, 60, 10047−10055

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amounts of RNA to DNA varies from 2 to 10 depending on the stage of cellular division,26 so Cu-2 could selectively target the 16s A-site rRNA over duplex DNA in vivo. To assess in vivo cleavage of the 16s RNA, total RNA was extracted from E. coli cells treated with each peptide. Analysis by agarose gel electrophoresis (Figure 6) shows that the 16s rRNA is not

Figure 6. Agaroge gel from total RNA extraction of E. coli cells treated with OV-3 derivatives. Lane 1, no complex; lane 2, Cu-GGH; lane 3, OV-3; lane 4, Cu-1; lane 5, Cu-2; lane 6, Cu-3; lane 7, Cu-4.

degraded and that the ratio of the 23s to 16s rRNA is around 2.2 for every peptide (Table 5). Therefore, intracellular targeting of the 16s rRNA is not a likely mechanism for the Cu-ATCUN OV-3 derivatives.

Figure 4. (a) Agarose gel shift assay for Cu-4 with Fl-16S rRNA and (b) a plot of % unbound Fl-16S A-site rRNA vs log[Cu-AMP] used to calculate binding affinity. Cu-AMPs were incubated with 1 μM Fl-16S rRNA up to a 60-fold excess and allowed to equilibrate for 1 h at room temperature before separation on an agarose gel. The intensity of the bands for the unbound Fl-16S rRNA was used to determine the KD value for each peptide.

Table 5. Ratio of 23s to 16s rRNA from E. coli Treated with OV-3 Derivatives peptide none Cu-GGH OV-3 Cu-1 Cu-2 Cu-3 Cu-4

ratio of 23s to 16s rRNA 2.6 2.8 2.6 2.5 2.4 3.0 2.1

± ± ± ± ± ± ±

0.3 0.8 0.5 0.5 0.3 0.5 0.4

Cellular Nuclease Activity. The terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay was utilized to determine if Cu-AMPs were capable of cleaving DNA in vivo. E. coli 25922 was treated with each Cu-AMP, and then the cells were subjected to the TUNEL reaction, in which the enzyme TdT incorporates a fluorescein-labeled dUTP at nicked sites in the bacterial genome. The number of fluorescently labeled cells was determined by flow cytometry, and the percentage is summarized in Table 4. Despite the high levels of plasmid cleavage from Cu-2 and Cu-4, these Cu-AMPs did not nick the DNA in vivo. These data indicate that nuclease activity does not contribute to the enhanced antimicrobial activity observed for Cu-2 and Cu-4 due to the absence of cleavage of RNA and DNA in vivo, but these peptides do have the potential to bind to intracellular targets, as well as a broader range of activities expressible through other targets. Neither Cu-1 nor Cu-3 displayed nuclease activity, most likely as a result of their lower α-helical character as determined by CD (Figure 1). Furthermore, both Cu-2 and Cu-4 are able to form dimers (Figure S12), and so these two metallopeptides are most likely able to more efficiently cleave DNA as a consequence of there being two metal binding domains. While it appears that these ATCUN

Figure 5. (a) Agarose gel from time-dependent degradation of Fl-16S rRNA by Cu-2 and (b) plots for [Fl-16S rRNA] vs time. The 100 nM Cu-AMPs were incubated with 1 μM Fl-16S rRNA, 1 mM ascorbate, and 1 mM hydrogen peroxide in 20 mM HEPES and 100 mM NaCl, pH 7.4. Reactions were monitored for up to 1 h at room temperature and then separated by agarose gel electrophoresis.

Although the binding affinity for the 16s A-site rRNA for Cu2 is about 3-fold weaker than its affinity for DNA, around 86% of RNA in the cell is rRNA.26 Furthermore, the relative 10051

DOI: 10.1021/acs.jmedchem.7b01117 J. Med. Chem. 2017, 60, 10047−10055

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structure in hydrophobic environments and the lowest MICs, this suggests that they are able to target membranes and induce membrane leakage. Ultrastructural damage by another cathelicidin peptide, SMAP-29, has been observed previously.1,3 Therefore, it appears that the addition of the Cu-ATCUN motif has altered the activity of the OV-3 binding domain for the sequences that do not contain the additional C-terminal GGC sequence. Lipid Peroxidation. The extent to which each metallopeptide was able to oxidize lipids was monitored via a lipid peroxidation assay. Malondialdehyde (MDA) is the most common secondary lipid peroxidation product produced as a result of decomposition of arachidonic and other poly unsaturated fatty acids, and the MDA assay allows quantification of the amount of MDA produced from lipid oxidation.29 ROS generated from our ATCUN derivatives can result in the production of lipid hydroperoxides that can undergo oneelectron reduction to initiate and propagate the production of more lipid hydroperoxides. These intermediates can also undergo decomposition via oxygen-dependent oxidative mechanisms to form MDA. E. coli 25922 was grown to the mid logarithmic phase and incubated with each peptide in the presence of hydrogen peroxide and ascorbic acid. Controls containing no peptide or coreagents, only coreagents, and CuCl2 were included. The cells were then harvested and treated with phosphoric acid and butylated hydroxytoluene to prevent additional oxidation of the lipids. Thiobarbituric acid (TBA) was then added to the reaction mix, and the solution was heated at 90 °C to form the (TBA)2-MDA adduct, which has a maximum absorbance at 532 nm. The reaction mixture was separated by reverse-phase (RP) HPLC, and the (TBA)2-MDA adduct eluted at around 11 min (Figure 7). Malondialdehyde was used to generate a standard curve in order to determine the amount of adduct formed by each Cu-AMP (Figure S11). E. coli 25922 incubated with Cu-GGH, OV-3, or ascorbate and hydrogen peroxide without complex showed background levels of lipid peroxidation while all four Cu-ATCUN derivatives of OV-3 displayed high levels of lipid peroxidation (Figure 7). These results demonstrate that both the OV-3 binding domain and the GGH metal binding domain are

derivatives do not exhibit an efficient nuclease function to kill bacteria, they do have the potential to target multiple cellular biomolecules as indicated by their in vitro binding and activity toward plasmid DNA and the 16S A-site rRNA. Multiple studies have implicated metal derivatives of AMPs to interact with and cleave nucleic acids, including Cu-ATCUN derivatives of buforin,17,27 WRWYCR,28 and histatin analogues.15 From these studies, it is possible to modify the sequence of our CuAMP derivatives to accumulate within the cell and specifically target nucleic acids. Membrane Permeabilization. The degree to which the Cu-AMPs were able to permeabilize the bacterial membrane was investigated by a β-galactosidase leakage assay. E. coli 25922 was grown to the mid logarithmic phase and βgalactosidase expression was induced with IPTG. Cells were then incubated with 32 μM Cu-AMP and grown for 1 h. Following centrifugation, the supernatant was mixed with 0.8 mg/mL of o-nitrophenyl-β-galactopyranoside (ONPG) and the absorbance was monitored at 405 nm for 1 h. Final absorbance readings are displayed in Table 6. While Cu-GGH, Cu-1, and Table 6. Summary of the Final Absorbance Reading at 405 nm for the β-Galactosidase Leakage Assaya complex CuCl2 Cu-GGH OV-3 Cu-1 Cu-2 Cu-3 Cu-4

A405 0.12 0.2 0.84 0.06 1.09 0.08 1.76

± ± ± ± ± ± ±

0.01 0.19 0.07 0.01 0.35 0.01 0.41

a

E. coli 25922 was grown to the mid logarithmic phase, induced with 1 mM IPTG for 1 h, and then incubated with 32 μM Cu-AMPs for 1 h. The supernatant was transferred to a 96-well plate and incubated with 0.8 mg/mL of ONPG, and the absorbance was monitored at 405 nm for 1 h.

Cu-3 showed no membrane leakage, Cu-2 and Cu-4 derivatives displayed high levels of membrane leakage. OV-3 was also able to disrupt the membrane, with a similar level of permeabilization as Cu-2. Since these same peptides have the most defined

Figure 7. (a) E. coli 25922 was grown to the mid log phase and then incubated with 10 μM Cu-AMPs with 1 mM ascorbate and 1 mM H2O2 for 2 h. The cells were then harvested and treated with 2-thiobarbituric acid (TBA) and malondialdehyde (MDA) to produce the (TBA)2-MDA adduct. The reactions were separated by HPLC to detect the chromophore, which elutes at 11 min. (b) Summary of lipid peroxidation data obtained for OV-3 derivatives. 10052

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column from Phenomenex. A gradient of 1%/min from water to acetonitrile (both containing 0.1% TFA) was used to elute the peptides to ≥95% purity and identity confirmed by ESI-MS. Determination of Peptide Concentration. The concentration of OV-3 was determined by creating a calibration curve of a known peptide using ESI mass spectrometry. The concentration of GGHRRWKIVVIRWRRGGC-NH2 was determined by UV−vis spectroscopy at 280 nm with an extinction coefficient of 5690 M−1 cm−1. Known amounts of the peptide were injected into the mass spectrometer, and the total intensity for the peaks from the peptide was used to generate a calibration curve to determine the concentration of OV-3 (Figure S3). The concentrations of peptides 1, 2, 3, and 4 were determined by performing a titration with NiCl2 and by monitoring the absorbance at 240 nm. The absorbance at 240 nm was plotted against the concentration of NiCl2, and the data was fit to a quadratic one-site binding equation to determine the concentration of the peptide. For all assays, the peptides were mixed with CuCl2 in a 1.1:1 ratio in 20 mM HEPES and 100 mM NaCl with a pH of 7.4. The Cu-peptide was allowed to equilibrate at room temperature for 30 min prior to use. Circular Dichroism. Circular dichroism (CD) was performed on a Jasco J-810 CD spectropolarimeter with 100 μM peptides in 750 μM phosphate buffer, pH 7.4, and 10 mM SDS in 10 mM Tris, pH 7.4. A total of three scans were taken for each sample, and the scans were smoothed. The percent β-sheet and α-helical content of each peptide was determined by application of the K2D3 program for each CD spectrum between 240 and 190 nm. Antimicrobial Assays. The antimicrobial activity of the peptides was tested against E. coli 25922, MRSA 43300, P. aeruginosa PAO1, K. pneumoniae, A. baumannii, S. pneumoniae, and E. faecium using a microdilution method. Serial dilutions (2×) of the peptides were prepared in 96-well plates beginning with 16 μM of each Cu-AMP. A negative control containing only CAMHB media and a positive control containing bacteria with no peptide were included in each row of the well plate. Cu-AMPs were incubated with bacteria containing (1−7) × 105 CFU/mL overnight in CAMHB media at 37 °C. The minimal inhibitory concentration (MIC) is the concentration of peptide that prevented growth of the bacteria. All experiments were conducted in triplicate with at least two independent trials for each. Plasmid DNA Isolation. PUC19 plasmid DNA was isolated from pUC19 transformed DH5α E. coli cell line and purified using the Qiagen Maxi prep kit. The plasmid was eluted in a buffer containing 20 mM HEPES and 100 mM NaCl, pH 7.4. The isolated DNA was quantified by UV−vis spectrophotometry using the absorbance at 260 nm. Time-Dependent Activity with pUC19. A DNA plasmid cleavage assay was utilized to assess the ability of Cu-AMPs to mediate DNA cleavage. Ten μL reactions were prepared and run in parallel with staggered start times at the following concentrations: 10 μM bp pUC19, 100 nM Cu-AMP, 1 mM ascorbate, and 1 mM hydrogen peroxide in 20 mM HEPES and 100 mM NaCl, pH 7.4. Data points were collected at 0, 2, 5, 10, 15, 20, 30, 45, and 60 min, and control reactions containing no complex and CuCl2 were also performed. Reactions were quenched by the addition of 2 μL of 6× loading dye containing 10 mM EDTA and separated by 1% agarose gel electrophoresis. A control lane was included on each gel to ensure that degradation was mediated by the complexes rather than exogenous degradation of the DNA plasmid. Gels were imaged with a BioRad gel doc instrument. The intensity of supercoiled, nicked, and linear bands was quantified using the GelQuantNET software, and a correction factor of 1.47 was applied to the intensity of the supercoiled bands to account for the limited ability of GelRed to intercalate with supercoiled DNA. The fraction of the total intensity observed in a band plasmid was used to calculate the concentration of supercoiled, S, nicked, N, and linear, L, DNA at each time point. From this data, firstorder rate constants for the rate of DNA nicking, k1, and linearization, k2, were determined from a first-order consecutive model with eqs 2−4. All experiments were conducted in triplicate with at least two independent trials for each.

required for lipid peroxidation. Similar to Cu-2 and Cu-4, ATCUN derivatives of anoplin are also able to promote lipid peroxidation in bacteria, resulting in enhanced bioactivity, and these peptides do not result in an increased membrane leakage.16 The addition of the ATCUN motif and C-terminal GGC sequence for our derivatives appears to increase the αhelical content of the peptides, which may promote tighter binding to the lipid bilayer thus resulting in greater membrane leakage and lipid peroxidation. In contrast to Cu-2 and Cu-4, neither Cu-1 nor Cu-3 showed an enhanced antimicrobial activity or permeabilized the membrane, most likely resulting from lower α-helical character in hydrophobic environments and an inability to form pores in the membrane. However, all of the metallopeptides were able to promote oxidative damage toward lipids, relative to the OV-3 targeting domain that was able to inhibit by an induced membrane leakage, but not promote lipid oxidation. Accordingly, Cu-2 and Cu-4 promote enhanced antimicrobial activity as a result of their ability to both permeabilize the plasma membrane and oxidize lipids.

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CONCLUSION In summary, we have demonstrated that Cu-ATCUN derivatives of OV-3 with a C-terminal GGC sequence display an increased antimicrobial activity toward multiple microorganisms. These peptides form an α-helix in lipids and SDS micelles and display an increased secondary structure relative to OV-3. They also show an increased membrane permeabilization and lipid peroxidation compared to OV-3 and Cu-GGH. Peptides containing the ATCUN motif lacking the GGC sequence were able to promote the oxidation of lipids, but did not permeabilize the membrane, most likely a reflection of their decreased α-helical character and resulting in MICs similar to OV-3, but mediated by a different mode of action. Taken together, these data suggest a model in which the Cu-ATCUN AMP targets the bacterial membrane through the OV-3 targeting domain. The Cu-GGH metal binding domain is then able to induce damage to the membrane, causing membrane leakage that results in bacterial death at a lower MIC relative to OV-3. This study demonstrates that CuATCUN derivatives of AMPs could provide a novel approach to developing new antibiotics and modifying the mode of action of currently used antibiotics.

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EXPERIMENTAL SECTION

Materials. Tris, EDTA, NaCl, ONPG, glycerol, xylene cyanol, bromophenol blue, 30% hydrogen peroxide with inhibitor, butylated hydroxytoluene, phosphoric acid, thiobarbituric acid, methanol, potassium phosphate monobasic, and calcium chloride were all purchased from Sigma-Aldrich. GelRed, ascorbic acid, yeast extract, and magnesium chloride were purchased from Fisher Scientific. HEPES and copper(II) chloride were purchased from J.T. Baker Co. The malondialdehyde standard was purchased from Cayman Chemical; tryptone was purchased from Teknova, and Mueller− Hinton broth was purchased from BD. IPTG was purchased from Gold Biotechnology. POPG and POPC lipids were purchased from Avanti. All amino acids used for peptide synthesis were from AAPPTec. Fluorescein-labeled 16S A-site rRNA with the sequence 5′-fluorescein-GGCGUCACACCUUCGGGUGAAGUCGCC-3′ was purchased from Dharmacon. Values reported from each experiment represent the average of three independent trials. Peptide Synthesis. Peptide 2 was purchased from AAPPTec. OV3, peptide 1, peptide 3, and peptide 4 were synthesized by standard solid-phase peptide synthesis using Fmoc chemistry on a PS3 synthesizer from Protein Technologies. The peptides were then purified via RP-HPLC with a C18 Gemini 5 μM 100 × 21.2 mm 10053

DOI: 10.1021/acs.jmedchem.7b01117 J. Med. Chem. 2017, 60, 10047−10055

Journal of Medicinal Chemistry

Article

β-Galactosidase Leakage Assays. E. coli 25922 was grown to an OD600 of 0.6 in LB media. IPTG was then added to 1 mM, and the cells were incubated for 1 h at 37 °C before washing three times with a PBS buffer and then resuspending in LB media. A 75 μL volume of resuspended cells were incubated with 75 μL of 64 μM Cu-AMP for 1 h at 37 °C. The cells were then spun down at 4500 rpm for 10 min, and 100 μL of the supernatant was loaded onto a 96-well plate. A 50 μL volume of 2.4 mg/mL of ONPG was added to each well, and the absorbance at 405 nm was monitored for 1 h at room temperature. A plot of absorbance vs time was used to calculate the rate of ONPG cleavage, and the final absorbance at 405 nm was recorded. All experiments were conducted in triplicate with at least two independent trials for each. Lipid Peroxidation Assays. E. coli 25922 was grown to an OD600 of 0.6. The cells were spun down and washed twice with a buffer (20 mM HEPES and 100 mM NaCl, pH 7.4) and then resuspended in a buffer. A 10 mL volume of cells was incubated for 2 h at 37 °C with 10 μM Cu-AMP, 1 mM ascorbate, and 1 mM H2O2. The cells were then spun down and resuspended in 750 μL of 0.44 M H3PO4, to which a 25 μL volume of 9.08 mM butylated hydroxytoluene (BHT) was added, and the reactions were incubated at room temperature for 10 min. A 250 μL volume of 41.6 mM 2-thiobarbituric acid (TBA) was then added, and the reactions were incubated at 90 °C for 30 min. After cooling to room temperature, the reaction mixture was separated by HPLC on a C18 column using an isocratic gradient of 65% 50 mM KH2PO4, pH 7.0, and 35% MeOH and was monitored at 532 nm. The peak that elutes at around 11 min corresponds to the malondialdehyde-TBA adduct, and a standard curve was used to determine the amount of lipid oxidation for each Cu-AMP. Controls containing no peptide and CuCl2 were also included. All experiments were conducted in triplicate with at least two independent trials for each.

Freifelder−Trumbo Analysis. The fractions of supercoiled ( fsup) and linear ( f lin) pUC19 plasmid were determined for the last three time points for each metallopeptide from the time-dependent reactions. These fractions are related to the number of single-stranded (n1) and double-stranded breaks (n2) defined by the Poisson distribution (eq 5) and the Freifelder−Trumbo relation (eq 6). Eqs 5 and 6 were used to calculate n2 and n1, respectively, and the ratio of n2/n1 was determined. Time-Dependent Activity with Fl-16S rRNA. An RNA cleavage assay with fluorescein-labeled 16S rRNA was used to determine the metallopeptides’ ability to cleave RNA. Ten μL reactions were prepared and run in parallel with staggered start times with the following concentrations: 1 μM Fl-16S rRNA, 100 nM Cu-AMP, 1 mM ascorbate, and 1 mM hydrogen peroxide in 20 mM HEPES and 100 mM NaCl, pH 7.4. Data points were collected at 0, 2, 5, 10, 15, 20, 30, 45, and 60 min, and control reactions containing no complex or CuCl2 were also performed. Reactions were quenched by the addition of 10 μL of 2× loading dye containing 10 mM EDTA and heated to 90 °C for 5 min prior to separation on a 2% agarose gel. A control lane was included on each gel to ensure that degradation was promoted by the complexes rather than exogenous degradation of the RNA. Gels were imaged with a BioRad gel doc instrument. Gels were quantified using the GelQuantNET software, and the concentration of RNA present in each lane was determined by dividing the intensity of the band at time t by the intensity of the band at time 0, t0. Plots of [RNA] vs time were fit to an exponential decay function to yield rate constants for RNA cleavage. All experiments were conducted in triplicate. Gel Shift Assays. Ten μM aliquots of pUC19 or 1 μM Fl-16S rRNA were incubated with 0, 1, 2, 5, 10, 20, 30, 40, 50, and 60 μM CuAMP in 20 mM HEPES and 100 mM NaCl, pH 7.4, for 1 h at room temperature. Two μL of 6× native dye (20% glycerol, 10 mM TrisHCl, pH 7.5, 0.025% bromophenol blue, 0.025% xylene cyanol) was added to 10 μL of each reaction mixture. DNA reactions were then separated on a 1% agarose gel, and RNA reactions were separated using a 4% agarose gel in 0.5× TBE buffer. Gels were imaged by the use of a BioRad gel doc instrument, and the intensity of each band (supercoiled, nicked, and linear) was quantified using the GelQuantNET software. A correction factor of 1.47 was applied to the intensity of the supercoiled bands to account for the limited ability of GelRed to intercalate with supercoiled DNA. The fraction of the total intensity observed in a band plasmid was used to calculate the concentration of supercoiled, nicked, and linear DNA at each time point. Plots of [free pUC19] or [free Fl-16S rRNA] vs log [Cu-AMP] were fit to a logarithmic function to yield the KD value for each peptide. All experiments were conducted in triplicate. TUNEL Assay. E. coli 25922 was grown overnight, diluted to 4 × 107 cells, and then incubated with 10 μM Cu-AMPs for 2 h at 37 °C. The cells were pelleted by centrifugation, washed with a cold PBS buffer twice, and then fixed with freshly prepared 4% paraformaldehyde in a PBS buffer for 30 min at room temperature. Afterward, cells were pelleted, resuspended in 0.1% Triton-X 100, 0.1% sodium citrate in a PBS buffer, and incubated on ice for 2 min. The cell mass was then pelleted and resuspended in 50 μL of the TUNEL reaction mixture containing 45 μL of the labeling solution and 5 μL of the TdT enzyme, and the reaction was allowed to proceed for 1 h at 37 °C. Subsequently, the reaction mixtures were centrifuged, washed with a PBS buffer, and then counterstained with 100 μL of 10 μg/mL of propidium iodide for 10 min. Five μL of cells were diluted in 1 mL of a PBS buffer, and a total of 10 000 cells were counted by flow cytometry. E. coli cells with no treatment were used as a negative control, while E. coli treated with 10 μM CuCl2 and 1 mM hydrogen peroxide were used as a positive control. Total RNA Isolation. E. coli 25922 was grown to the mid logarithmic phase followed by incubation with 10 μM of each peptide for 2 h at 37 °C. The total RNA from 2 × 107 cells was harvested using the Absolutely RNA miniprep kit (Stratagene) according to the manufacturer’s instructions. Ten μL of the eluted RNA was loaded onto a 2% agarose gel prestained with GelRed. The bands corresponding to the 23s and 16s rRNA were quantified using the GelQuantNET software.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.7b01117. Chromatograms and mass spectra for each OV-3 derivative, calibration curve for determining OV-3 concentration, gels and plots for gel-shift assays and time-dependent cleavage assays for pUC19 and 16s rRNA with each peptide, and calibration curve for lipid peroxidation assay (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: 614 292 2703. Fax: 614 292 1685. ORCID

Zhen Yu: 0000-0003-2092-9960 J. A Cowan: 0000-0002-4686-6825 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by grants from the National Institutes of Health (HL093446). Z.Y. was supported by the Pelotonia Fellowship Program.

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ABBREVIATIONS USED ATCUN, amino terminal copper and nickel binding motif; OV, ovispirin; SMAP-29, sheep-myeloid antimicrobial peptide 29; AMP, antimicrobial peptide; MDA, malondialdehyde; TBA, thiobarbituric acid; BHT, butylated hydroxytoluene; TBE, tris10054

DOI: 10.1021/acs.jmedchem.7b01117 J. Med. Chem. 2017, 60, 10047−10055

Journal of Medicinal Chemistry

Article

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borate-EDTA; TdT, terminal deoxynucleotidyl transferase; PBS, phosphate buffered saline; RP-HPLC, reverse-phase HPLC; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling; ONPG, ortho-nitrophenyl-β-galactoside; CAMHB, cation-adjusted Mueller Hinton broth; MIC, minimal inhibitory concentration; LB, Luria−Bertani; IPTG, isopropyl β-D-1-thiogalactopyranoside.

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REFERENCES

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DOI: 10.1021/acs.jmedchem.7b01117 J. Med. Chem. 2017, 60, 10047−10055