Article pubs.acs.org/jmc

Antimicrobial Peptides with Potential for Biofilm Eradication: Synthesis and Structure Activity Relationship Studies of Battacin Peptides Gayan Heruka De Zoysa,† Alan James Cameron,† Veena V. Hegde,‡ Srinivasarao Raghothama,*,‡ and Vijayalekshmi Sarojini*,† †

School of Chemical Sciences, The University of Auckland, Private Bag, 92019 Auckland, New Zealand NMR Research Centre, Indian Institute of Science, Bangalore 560012, India

‡

S Supporting Information *

ABSTRACT: We report on the first chemical syntheses and structure−activity analyses of the cyclic lipopeptide battacin which revealed that conjugation of a shorter fatty acid, 4-methylhexanoic acid, and linearization of the peptide sequence improves antibacterial activity and reduces hemolysis of mouse blood cells. This surprising finding of higher potency in linear lipopeptides than their cyclic counterparts is economically beneficial. This novel lipopeptide was membrane lytic and exhibited antibiofilm activity against Pseudomonas aeruginosa, Staphylococcus aureus, and, for the first time, Pseudomonas syringe pv. actinidiae. The peptide was unstructured in aqueous buffer and dimyristoylphosphatidylcholine-polymerized diacetylene vesicles, with 12% helicity induced in 50% v/v of trifluoroethanol. Our results indicate that a well-defined secondary structure is not essential for the observed antibacterial activity of this novel lipopeptide. A truncated pentapeptide conjugated to 4-methyl hexanoic acid, having similar potency against Gram negative and Gram positive pathogens was identified through alanine scanning.

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INTRODUCTION

reported to have a better therapeutic index than polymyxin B. Synthesis of battacin has not been reported in the literature. This article focuses on the design, first chemical syntheses, and antibacterial activity analyses of a series of battacin analogues. The efficacy of four cyclic lipopeptides with different N-terminal fatty acids, their corresponding linear counterparts, and Fmoc protected battacin were evaluated, in comparison to synthetic battacin, against different bacterial pathogens. Timecourse killing, antibiofilm testing, membrane permeability assays, scanning electron microscopy, and solution conformational analysis of the most potent analogue has been undertaken. Amino acid residues crucial for antimicrobial activity were identified through combinatorial alanine scanning, resulting in a truncated version that retained similar potency.

In contrast to naturally occurring antimicrobial peptides (AMPs) which are genetically encoded and synthesized on the ribosome, naturally occurring lipopeptides are a class of nonribosomally synthesized, structurally diverse antibiotics with their N-termini conjugated to long chain fatty acids.1 Both AMPs and lipopeptides hold promise as future broad spectrum antimicrobial agents, particularly in the context of the alarming increase in the emergence of multidrug resistant (MDR) microbial pathogens. The lipopeptide antibiotics daptomycin and polymyxin B, produced by soil bacteria, are used in the treatment of methicillin resistant Staphylococcus aureus (MRSA) infections.2−4 However, polymyxin B is only used as the last line of defense for otherwise untreatable serious infections, particularly because of nephrotoxicity concerns. This, together with the emergence of resistance to polymyxin B, highlights the need for short and structurally simple peptides with broad spectrum antimicrobial activity accessible in a cost-effective manner. Battacin is a novel cyclic lipopeptide, produced by the soil isolate Paneibacillus tianmunesis and belongs to the octapeptin group of peptide antibiotics which are characterized by a high percentage of the nonprotein amino acid α,γdiaminobutyric acid (Dab) and a branched β-hydroxy fatty acid tail linked to a cyclic heptapeptide moiety.5,6 Battacin has been © 2014 American Chemical Society

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RESULTS AND DISCUSSION

Fatty Acid and Peptide Synthesis. The fatty acid moiety 6 ((3RS,6RS)-3-hydroxy-6-methyl-octanoic acid) was synthesized in five steps from commercially available 4-methyl hexanoic acid (1) following a literature procedure reported by Sakura et al. (Scheme 1).7 The hydroxyl group of 6 was left unprotected while conjugating to the peptide as Sakura et al. Received: July 17, 2014 Published: December 15, 2014 625

DOI: 10.1021/jm501084q J. Med. Chem. 2015, 58, 625−639

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Journal of Medicinal Chemistry

in DCM for 1 h to generate linear version of battacin (8) with protected side chains, except for Dab2. The side chain NH2 of Dab2 was coupled to COOH of Leu8 using a mixture of 3 equiv of TBTU/HOBt and 1% v/v of DIPEA at high peptide dilution (1.5 mM) for 3 h in DMF to produce 9 (the protected cyclic lipopeptide). On completion of cyclization at the end of 3 h, as confirmed by HPLC, the remaining protecting groups were removed from 9 using TFA-cocktail following normal protocols to yield 10 (battacin) (Scheme 2) in 33% overall yield, as a mixture of isomers as in the case of the isolated natural product. Syntheses and activity studies of the four stereoisomers of battacin is currently in progress in our laboratory and will be reported in future. The same protocol was followed for the synthesis of all cyclic lipopeptide analogues. Linear lipopeptides were synthesized on Tentagel S-NH2 resin as described under materials and methods. Analytical HPLC traces, ESI-MS, and 1 H NMR spectra of all peptides are provided in the Supporting Information. Rationale Behind the Choice of Battacin Analogues. Battacin and polymyxin B have very similar amino acid compositions, both having five α,γ-diaminobutyric acid residues, one D -Phe and one leucine. The fatty acid components in both peptides are of similar length (nine carbons in battacin and eight in polymyxin B (Figure 1).5,11,12 Interplay of the hydrophobic domains (acyl chain and D-PheLeu) and the five cationic Dab residues of polymyxin B have been shown to be important for antibacterial activity and lipopolysaccharide binding.12−14 To gain insight into the structure−activity relationship of battacin, we designed nine analogues (Table 1). The antibacterial activity of lipopeptides can be sensitive to the Nterminal fatty acid component with longer fatty acids expected to enhance membrane interaction, potentially leading to enhancement of antibacterial activity. Three different cyclic

Scheme 1. Synthesis of 6 ((3RS,6RS)-3-hydroxy-6methyloctanoic Acid)a

a

Reagents and conditions: (a) SOCl2 (2.27 equiv), reflux, 5 h (1 g, 95%); (b) ethyl acetoacetate (2 equiv), sodium (1 equiv) in dry diethyl ether under N2, into which 2 was added and refluxed for 24 h (1.8 g, 52%); (c) NaOH (1 equiv), 100 °C, 45 min (2.18 g, 54%); (d) NaBH4 (0.5 equiv), ethanol, 2 h (0.55 g, 50%); (e) KOH (2 equiv), ethanol, reflux, 1.5 h (0.3 g, 64%).

has successfully managed to couple the unprotected fatty acid in a similar lipopeptide. All other fatty acids were commercially obtained and used directly for on-resin acylation reactions. The peptide components of all cyclic lipopeptides were assembled on 2chlorotrityl chloride resin as this linker enables peptide−resin cleavage under mild acidic conditions generating suitably protected peptides, essential for the subsequent solution phase cyclization step.8,9 Conjugation of the fatty acids was carried out on-resin following the normal peptide coupling procedure. In naturally occurring battacin, the side chain of Dab2 forms a peptide bond to the COOH group of C-terminal Leu, resulting in a cyclic structure. To permit selective deprotection, the side chain of Dab2 was protected as the acid sensitive 4-methyltrityl (Mtt) derivative.10 Selective removal of Mtt and simultaneous cleavage of peptide (7) from the resin was achieved using 20% trifluoroethanol (TFE) Scheme 2. Solid Phase Synthesis of 10a

a

Reagents and conditions: (a) Fmoc-Leu-OH (4 equiv), DIPEA (5 equiv) in DCM for 1 h; (b) Fmoc amino acid (4 equiv), TBTU (3.9 equiv), HOBt (3.9 equiv), DIPEA (10 equiv) in DMF 45 min, deprotection 20% piperidine in DMF 20 min; (c) 20% TFE in DCM, 1 h; (d) TBTU (3 equiv), HOBt (3 equiv), DIPEA (1% v/v in DMF), 3 h; (e) TFA−TIS−H2O (95:2.5:2.5 (v/v/v) 3 h. 626

DOI: 10.1021/jm501084q J. Med. Chem. 2015, 58, 625−639

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Journal of Medicinal Chemistry

Figure 1. Amino acid sequences and chemical structures of battacin and polymyxin B* *FA: fatty acid.

Table 1. Amino Acid Sequences of the Peptides Studied

peptide

sequence

12 13 14 15 16 17 18 19 20

Fmoc-D-Dab-cyclo[Dab-Dab-Leu-D-Phe-Dab-Dab-Leu] R1-D-Dab-cyclo[Dab-Dab-Leu-D-Phe-Dab-Dab-Leu] R2-D-Dab-cyclo[Dab-Dab-Leu-D-Phe-Dab-Dab-Leu] R3-D-Dab-cyclo[Dab-Dab-Leu-D-Phe-Dab-Dab-Leu] R3-D-Lys-cyclo[Lys-Lys-Leu-D-Phe-Lys-Lys-Leu] R1-D-Dab-Dab-Dab-Leu-D-Phe-Dab-Dab-Leu-NH2 R2-D-Dab-Dab-Dab-Leu-D-Phe-Dab-Dab-Leu-NH2 R3-D-Dab-Dab-Dab-Leu-D-Phe-Dab-Dab-Leu-NH2 R3-D-Lys-Lys-Lys-Leu-D-Phe-Lys-Lys-Leu-NH2

Table 2. Antibacterial Activity of Synthetic Analogues of Battacina MIC (μM) Ea1501

Str4Ea

E. coli

Psa 16207

P. aeruginosa

S. aureus

10

24−49

24−49

10−15

5−10

5−10

>50

cyclic

12 13 14 15 16

19−32 15−25 98−488 92−461 81−409

32−65 25−50 488−976 23−46 20−41

19−32 15−25 98−488 92−461 81−409

19−32 5−10 49−98 46−92 12−20

2−5 2.5−5 10−15 92−461 409−817

2−5 101−507 98−488 92−461 81−409

linear

17 18 19 20

2.5−5 24−48 9−14 12−21

5−10 48−96 14−23 12−20

2.5−5 24−48 5−9 12−20

1−2.5 14−24 14−23 12−20

1−2.5 48−96 14−23 12−20

1−2.5 48−96 5−9 1−2.5

antibiotic controls

streptomycin gentamycin

1−2.5 N

500−1000 N

1−2.5 N

1−2.5 N

N 3 log difference in comparison to the control) within 3 h of incubation with 17 at 20× MIC. At 2× and 4× MICs, up to 9 h, >3 log difference in bacterial growth, in comparison to the control (no peptide), was observed confirming the bactericidal nature 17 at these concentrations.18 However, at 24 h, significant regrowth was observed at 2× and 4× MICs, indicating that the peptide is bacteriostatic at these concentrations. In comparison, slower killing kinetics were observed against E. amylovora. We believe it is because of the slow growing nature of this bacterium rather than the antibacterial nature of the peptide. Ea1501 did not grow in the presence or absence of the peptide during the initial 6 h of incubation. Apart from at 1× MIC, which regrew after 15 h of incubation, complete killing of Ea1501 was observed after 9 h of incubation with 17 at 2×, 4×, and 20× MIC, indicating that the peptide is bactericidal against Ea1501 at these concentrations. The peptide showed rapid action against the kiwi fruit pathogen, Psa16027. Complete killing of Psa was observed 629

DOI: 10.1021/jm501084q J. Med. Chem. 2015, 58, 625−639

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Figure 4. Effect of treating Pseudomonas aeruginosa biofilms with 17 at various concentrations for 48 h. The lipopeptide was present at 10 (b,f), 50 (c,g), and 100 μM (d,h) concentrations, respectively. (b−d) Effect on initial biofilm formation (lipopeptide added at incubation). (f−h) Effect on eradication of preformed biofilms (lipopeptide added to preformed biofilms. (a,e) Controls with no lipopeptide added. Bar = 20 μm.

Figure 5. Effect of treating preformed biofilms of Staphylococcus aureus with the 17 at various concentrations for 24 (a−e) and 48 (f−j) h. (The lipopeptide was present at 10 (b,g), 25 (c,h), 50 (d,i), and 100 μM (e,j) concentrations, respectively. (a,f) Controls with no peptide added. Bar = 20 μm.

630

DOI: 10.1021/jm501084q J. Med. Chem. 2015, 58, 625−639

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Journal of Medicinal Chemistry aeruginosa, and S. aureus. P. aeruginosa is an opportunistic human pathogen that forms biofilms and colonizes the lung tissues in cystic fibrosis patients with no satisfactory cure currently available.20 Psa is an economically important horticultural pathogen and has been reported to form biofilms inside and outside of the affected kiwi fruit plants.21,22 Activity against Psa biofilms is yet to be reported. S. aureus was included in our study as a model Gram positive human pathogen. The ability of the peptide to prevent the formation of biofilms and eradicate preformed biofilms of Psa was investigated using a crystal violet staining experiment. A semiquantitative estimate of the attached biofilm biomass of Psa16027 grown for 48 h in the presence and absence of the peptide is shown in Figure 3. Results indicate that 17 prevented initial attachment of Psa biofilms at concentrations as low as 5 μM (Figure 3a). Additionally, a 24 h treatment with the peptide resulted in complete eradication of the preformed biofilms of Psa (formed for 48 h) at peptide concentrations between 5 and 10 μM (Figure 3b). Microscopic images of crystal violet stained biofilms at 48 h treated with different concentrations of the peptide added at incubation and on preformed biofilms (Figure S2, Supporting Information) clearly revealed a dose dependent effect of the lipopeptide on Psa biofilms. The lipopeptide also inhibited initial biofilm formation and eradicated preformed biofilms of the human pathogens P. aeruginosa between 10 and 50 μM and S. aureus between 10 and 25 μM (Figures 4, 5 and Figure S3, Supporting Information). Bacteria within biofilms are protected by extracellular polymer matrices (EPS) that are impervious to antibiotics, which is one of the major factors that cause antibiotic resistance. The ability to penetrate the EPS matrix of biofilms will be essential for treating chronic infections caused by bacterial biofilms. The observed dose dependent effect of the lipopeptide 17 on these bacterial biofilms could be indicative of the lipopeptide penetrating the EPS matrix barrier. Membrane Permeabilization Studies. The membrane lytic property of the lipopeptide was elucidated in detail using dyes that specifically stain outer and inner bacterial membranes as well as monitoring morphological changes to the bacterial cells using scanning electron microscopy as described below. Outer Membrane Permeability. N-Phenylnaphthylamine (NPN) is a chemical dye which fluoresces weakly in an aqueous environment but strongly in a hydrophobic environment such as the outer bacterial membrane.23 The addition of 17 to P. aeruginosa suspensions in the presence of NPN caused an increase in fluorescence intensity, within 1 min, which plateaued at 32 μM lipopeptide concentration (Figure 6a), with no further increase in fluorescence intensity observed up to 100 μM, indicating that all the available surface area of the cell membrane has been permeablized by the lipopeptide at 32 μM. Inner Membrane Permeability. The ability of 17 to permeabilize the inner membranes of P. aeruginosa and S. aureus was evaluated using the propodium iodide uptake assay. Upon binding to DNA, propidium iodide fluoresces strongly, which is indicative of a compromised inner membrane integrity.24 Increase in PI fluorescence observed (Figure 6b) with increasing lipopeptide concentrations upon incubation with P. aeruginosa and S. aureus indicates the ability of 17 to disrupt the cytoplasmic membranes of these bacteria, resulting in the diffusion of PI into the bacterial cytoplasm. It is known in the literature that polymyxin B (PMB) binds to the anionic lipopolysaccharides (LPS) on the surface of the outer bacterial

Figure 6. (a) Outer membrane permeabilization assessment using Nphenylnaphthylamine (NPN) uptake assay on P. aeruginosa. (b) Inner membrane permeabilization assessment using propidium iodide on P. aeruginosa and S. aureus. * Indicates P < 0.05.

membranes and causes self-prompted uptake across the outer membrane, eventually reaching the periplasmic space and interacting with the cytoplasmic membrane.25 The NPN and PI uptake assays confirm that 17 is membrane lytic and has a similar mechanism of action to PMB. Scanning Electron Microscopy. The effect of 17 on Gram negative and Gram positive bacterial cell morphology was also studied using scanning electron microscopy. Untreated bacterial cells were rod shaped for Gram negative bacteria (Psa, P. aeruginosa and E. coli) and spherical in shape for the Gram positive pathogen (S. aureus), with all cells showing smooth intact surfaces (Figure 7). Treatment with 17 at 2× MIC led to severely perturbed cell membranes in all cases, leading to corrugated, blistered appearance. In most cases, complete lysis was observed with cellular remnants scattered across. Comparison with the untreated control cells clearly indicates that the lipopeptide caused severe damage to the cell membranes of the pathogens investigated and confirm its membrane permeabilizing ability. These symptoms are similar to the membrane lytic activity demonstrated by antimicrobial peptides against clinically relevant pathogens.26 Solution Conformation of 17. The CD spectra of 17 in aqueous and membrane mimetic environments (Figure 8) suggest that the peptide adopts a random coil conformation in water which shifts toward a more ordered (helical) state in 50% TFE. The single minimum in the CD spectrum at 200 nm decreases in intensity, and shifts to 205 nm in 50% TFE, with the appearance of a second minimum at 222 nm. Decrease in intensity and a slight shift toward higher frequency of the 200 nm band is also observed in methanol, which is indicative of a small structural change in this solvent. The shifts to the bands 631

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Journal of Medicinal Chemistry

showed a transition from random structure in aqueous buffer to a more ordered structure with increasing concentrations of TFE.27 Despite the lack of an ordered conformation in DMPC:PDA vesicles, the SEM images and the membrane permeability assays described above confirm that 17 causes membrane lysis implying that a well-ordered secondary structure is not essential for the observed antimicrobial activity of this peptide. NMR Studies. The 1H NMR spectra of 17 were recorded in CD3OH as well as a mixture of 70% water and 30% TFE (Supporting Information). Sequence specific assignments were obtained using a combination of ROESY and TOCSY spectra (Supporting Information). The ROESY spectrum of the peptide in methanol showed relatively weaker sequential NiH ↔ Ni+1H NOEs over the entire length of the peptide. Comparatively, the sequential CαiH↔Ni+1H NOEs were stronger (Figure S6, Supporting Information). The observed NOE pattern is indicative of no definite secondary structure for the lipopeptide in CD3OH and is in agreement with the CD spectrum in methanol. Lack of NOEs to the N-terminal alkyl chain is indicative of extensive molecular motion of the Nterminus. The rather large (∼6 ppb/K) temperature coefficients (dδ/dT) of most amide protons and coupling constants (3JNHCαH) ≥ 7 Hz (Table 3) indicate the solvent exposed nature of these protons. The relatively lower dδ/dt (80% crude purity following the same protocols discussed in the manuscript and assayed these against the pathogens under study. Bioassay results obtained using the synphase lantern peptides were in agreement with those obtained from the individually synthesized peptides. Results indicate that Leu8, Dab7, and Dab2 are not critical for the observed antimicrobial activity of 17 as replacement of these amino acids with alanine did not lead to significant 633

DOI: 10.1021/jm501084q J. Med. Chem. 2015, 58, 625−639

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residues and the D-Dab at position 1 are crucial for the observed antibacterial activity against the tested pathogens. This core pentapeptide 30 (Figure 10) was as potent as 17 against P. aeruginosa, S. aureus, and Psa.

Table 5. MIC (μM) of Alanine Scan Analogues of 17 MIC (μM) analogue

P. aeruginosa

S. aureus

E. coli

Psa 16207

17 21 22 23 24 25 26 27 28 29 30 (GZ3.159)

1−2.5 5−10 5−10 50−100 >1000 >1000 10−15 2.5−5 25−50 2.5−5 1−2.5

1−2.5 5−10 5−10 500−1000 25−50 >1000 25−50 2.5−5 >1000 >1000 2.5−5

2.5−5 15−25 5−10 100−500 25−50 500−1000 25−50 25−50 500−1000 100−500 10−15

1−2.5 10−15 2.5−5 100−500 >1000 >1000 25−50 2.5−5 100−500 5−10 1−2.5

Figure 10. Structure of the active pentapeptide moiety.

Hemolysis of Mouse Blood Cells. For an antimicrobial peptide to be a realistic prospect as a pharmaceutical agent, promising antimicrobial activity must be combined with low cytotoxicity against mammalian cells. One drawback of cationic AMPs is the higher levels of hemolytic activity associated with them. The hemolytic activity of the most potent lipopeptides from this study, 12, 13, 17, 19, 29 (devoid of the fatty acid), and the core pentapeptide 30, to mouse red blood cells was assessed to gain further insight into their potential as future pharmaceuticals. Percentage of hemolysis observed was negligible (95% purity as determined by analytical RP-HPLC performed using a Phenomenex Luna 5 μm C18 100 Å (250 mm × 4.6 mm) column using the same solvent system as above at flow rate of 1 mL per min. The identity of the peptides was established using electrospray ionization mass spectrometry (ESIMS) recorded on a Bruker micrOTOFQ mass spectrometer. Synthesis and Purification of Cyclic Lipopeptides. The peptides were assembled on acid sensitive 2-chlorotrityl chloride resin with a substitution level of 0.8−1.5 mmol/g at 0.1 mmol scale using the same coupling conditions described above. The side chain of Dab at position two was protected with the acid sensitive protecting group 4-methyltrityl (Mtt).10 On completion of chain assembly and Nterminal acylation, the side chain protected (except for Dab-2) linear lipopeptides were cleaved from the resin under mild acidic conditions using 20% 2,2,2-trifluoroethanol in DCM for 1 h.8,9 The cleavage mixture was evaporated under reduced pressure, the crude peptides precipitated by the addition of cold diethyl ether, the residue resuspended in cold ether, centrifuged three times, and the residue lyophilized to yield the peptides as white fluffy solids. Peptide cyclization was carried out in DMF at 1.5 mM concentration using 3fold excess of TBTU, HOBt, and 1% DIPEA for 3 h.42 The reaction mixture was concentrated under reduced pressure, dissolved in water, and lyophilized to yield the protected cyclic lipopeptides as white fluffy solids. Global deprotection was carried out using 95% TFA and 2.5% each of TIS and water (v/v) for 3 h. The cleavage mixture was evaporated, under reduced pressure, dissolved in water, and lyophilized to obtain the crude cyclic lipopeptides which were purified to homogeneity using reversed phase HPLC (see above) and characterized using ESI mass spectra recorded on a Bruker micrOTOFQ mass spectrometer. All peptides were purified to homogeneity to >95% purity as determined by analytical RPRPHPLC. Physical characteristics of the peptides are listed in Table S1, Supporting Information. Analytical HPLC traces, ESIMS spectra, and 1H NMR of the purified linear and cyclic lipopeptides are provided under Supporting Information. Alanine Scanned Library Using D-Series Lanterns. D-Series synphase lanterns trityl alcohol (5 μmol) was treated with 5 mL solution of 10% (v/v) acetyl chloride in dry DCM at room temperature for 3 h to produce trityl chloride lanterns. The reagent solution was decanted and lanterns washed with DCM (3 × 3 min) and used immediately in the next reaction. A mixture of 120 mM Fmoc-amino acid and 240 mM DIPEA (0.21 mL) dissolved in DMF (15 mL) was added to the lanterns and allowed to react for 90 min. Lanterns sharing common amino acids at the same position were kept in the same reaction vessel, whereas the lanterns in which amino acids at specific positions were replaced with alanine were suspended in separate reaction vessels. All lanterns were pooled together and washed with DMF and methanol. The Fmoc group from the lanterns was removed by submerging the lanterns in 20% piperidine for 30 min followed by washing with DMF and methanol. The lanterns were sorted as required and the subsequent Fmoc amino acid (120 mM) coupled using 240 mM of TBTU and DIPEA for 90 min.34 Fmoc deprotection, coupling, and washing steps were repeated until the desired sequences were obtained. On completion of syntheses, the lanterns were thoroughly washed with DCM and dried under vacuum overnight. The lanterns were separated into eight reaction vessels and the peptides cleaved off the lanterns with TFA cocktail mixture (TFA− TIS−H2O - 95:2.5:2.5 v/v) for 3 h. The TFA mixtures were evaporated separately under stream of N2 and the peptides precipitated using excess cold diethyl ether. The peptides were centrifuged thrice, resulting in white pellets which were dissolved in water and lyophilized to recover the crude peptides as white fluffy

discussed in the paper, similar trends have been reported for polymyxin B nonapeptide (PMBN) and octapeptin.11,12 However, in contrast to polymyxin, linearization has been found to be beneficial to antibacterial activity in this series of peptides.37 This is encouraging for the development of clinically relevant antimicrobial peptides because it significantly simplifies the synthesis part and thereby reduces manufacturing cost. Activity against the Gram positive human pathogen, S. aureus, is an added bonus unlike in the case of polymyxins. The antimicrobial potency, lack of hemolysis up to 1 mM, ability to permeabilize bacterial membranes, and disperse preformed biofilms of Gram negative and Gram positive bacteria make 17 a lead molecule for further development as broad spectrum antimicrobial peptide. Identification of amino acid residues critical for activity led to the generation of a truncated peptide 30 with similar potency and negligible hemolysis, further minimizing the cost of synthesis. For the first time, we have demonstrated that short membrane lytic lipopeptides like 17 can inhibit biofilm formation and disperse preformed biofilms of the kiwi fruit pathogen Psa, probably by penetrating the extracellular polymer matrix. To conclude, this study has resulted in the unique and probably the first observation that linearization of a cyclic peptide sequence can be beneficial to antimicrobial activity. At the same time, our study also agrees with the literature observations on polymyxin B and octapeptin peptides, where the N-terminal fatty acids have been found to influence their biological activities. Our laboratory is currently evaluating the potential of 17 and the truncated pentapeptide 30 against clinically relevant pathogens, particularly ESBL producer strains of E. coli, Klebsiella pneumoniae, and methicillin resistant S. aureus and their immobilization on a silicone surface to further assess their potential as antimicrobial agents in clinical settings.

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EXPERIMENTAL SECTION

Chemicals and Reagents. Fmoc protected leucine and Dphenylalanine, 2-chlorotrityl chloride resin, and Fmoc-rink amide linker were purchased from GL Biochem (Shanghai, China). Fmocdiaminobutyric acid (Fmoc-Dab) was purchased from Alabiochem (China). 4-Methylhexanoic acid, trifluoroacetic acid, piperidine, and myristic acid were obtained from Sigma-Aldrich. Tentagel S NH2 resin was obtained from Peptide International (Louisville, USA). 2,2,2Trifluoroethanol and geranic acid were purchased from Alfa Aesar (England). Fatty Acid Synthesis. 3-Hydroxy-6-methylotanoic Acid (6). 3Hydroxy-6-methylotanoic acid (6) was synthesized according to literature procedures.7 HRMS-ESI (m/z): [M − H]− calculated for C9H17O3, 173.1178; observed, 173.1189. Synthesis and Purification of Linear Lipopeptides. All peptides were synthesized by manual solid phase synthesis using standard Fmoc protocols. The peptides were assembled on Tentagel S NH2 resin (substitution level of 0.29 mmol/g) as C-terminal amides using Rink amide linker on a 0.1 mmol scale. tert-Butyloxycarbonyl (Boc) was used as the side chain protecting group of Dab. Amino acid couplings were mediated in DMF solution using TBTU as the coupling reagent, HOBt, as the additive and DIPEA as the base. The same coupling procedure was used for coupling the fatty acids to the N-termini of the peptides. The final lipopeptides were cleaved from the resin using 10 mL of the TFA cocktail mixture (TFA−TIS−H2O: 95:2.5:2.5 v/v) per gram of the resin. The cleavage mixture was evaporated under reduced pressure and the crude peptides precipitated using a large excess of cold diethyl ether. The white solid was resuspended in cold ether, centrifuged thrice to result in a white pellet which was dissolved in water and lyophilized to recover the crude peptides as white fluffy solids. The crude peptides were purified to homogeneity using reversed-phase high performance liquid chroma635

DOI: 10.1021/jm501084q J. Med. Chem. 2015, 58, 625−639

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Journal of Medicinal Chemistry

μM concentrations were incubated with the Psa16027 culture (50 μL) and LB medium (900 μL) for 48 h, at which time the supernatant from each well was removed and the wells gently washed twice with sterile water and stained with 1% crystal violet for 5 min. The dye was removed by decantation, and the wells were gently rinsed twice with 1 mL of sterile water. The stained biofilms in individual wells were viewed at 400× magnification under the microscope (Nikon eclipse TE2000) Effect of 17 on Psa16027 Preformed Biofilms. The ability of 17 to disperse preformed biofilms of Psa16027 was also investigated by crystal violet staining in 96-well microtiter plates. After an initial 48 h of biofilm formation as described above, in the absence of test peptides, the supernatant from each well was carefully removed without damaging the preformed biofilm architecture. Each well was washed twice gently with sterile water (1 mL) and supplemented with 100 μL of fresh LB alone (control wells) or 100 μL of 1, 5, and 50 μM concentrations of 17 in sterile LB medium and incubated for a further 24 h at 28 °C. These treated preformed biofilms were then subjected to crystal violet staining, imaging, and semiquantitative estimation of the biofilm mass as described above. Inhibition of P. aeruginosa and S. aureus Biofilm Formation: Live/Dead Staining. Biofilms of P. aeruginosa and S. aureus were prepared in 12-well plates as described above using sufficient numbers of positive and negative controls and 17 at various concentrations. To provide a surface for biofilm formation which was amenable for microscopy at higher magnification, circular coverslips (15 mm diameter, 1 mm thick) were inserted into each well at the start of bacterial inoculation. 17 (50 μL) at 5, 10, 25, 50, and 100 μM concentrations were incubated with the diluted bacterial cultures (50 μL) and LB medium (900 μL) for 48 h. The coverslips were removed from the wells, rinsed with sterile water to remove any planktonic cells, and dried at 50 °C for 20 min. A solution of live/dead bacterial stain, BacLight (Invitrogen) was used to stain the biofilm. BacLight stain was prepared (3:1000 dilution) using 1.5 μL each of SYTO 9 (greenfluorescent nucleic acid stain) and propidium iodide (red fluorescent nucleic acid stain) solution in 997 μL of sterile water. Then 60 μL of this BacLight solution was dispensed onto each of the coverslips and incubated in the dark for 25 min at room temperature. After 25 min, the excess stain was washed off with sterile water and the coverslips were air-dried. The dried coverslips were inverted onto a drop of mounting oil (supplied with BacLight kit) on a fresh glass slide for examination of biofilms at 1000× magnification using a Nikon Eclipse E600 microscope. The stained biofilms were viewed separately using FITC 2 and Tx Red filters, and the images were captured using an inbuilt digital sight DS-U1 camera. The images were merged using the in-built ACT-2U software. Eradication of P. aeruginosa and S. aureus Preformed Biofilms: Live/Dead Staining. The ability of 17 to disperse preformed biofilms of P. aeruginosa and S. aureus was also investigated using a Live/Dead staining experiment. After an initial 48 h of biofilm formation as described above, but without the addition of the test peptides to the wells, the supernatant from each well was carefully removed without damaging the preformed biofilm architecture. Each well was washed twice with sterilized water (1 mL) and supplemented with 100 μL of 17 at 10, 50, and 100 μM concentrations and sterile LB medium and incubated for a further 24 h at 37 °C. The preformed biofilm was subjected to Live/Dead staining as described above. 1-N-Phenylnaphthylamine (NPN) Uptake Assay.44,45 P. aeruginosa strain was grown overnight in Luria broth at 37 °C. The overnight culture was incubated for another 3−6 h in fresh LB to obtain OD600 of 0.5. The bacterial cells were centrifuged at 4600 rpm for 10 min. The bacterial cells were washed thrice with 10 mM phosphate buffer (pH 7.0) before resuspension in the same buffer to obtain an OD600 of 0.5. Then, 2.94 mL of the bacterial suspension was added to various peptide concentrations (30 μL, 0.5−100 μM) followed by addition of 60 μL of 0.5 mM 1-N-phenylnaphthylamine (NPN) to attain a final NPN concentration of 10 μM. Florescence measurements of the samples were performed at excitation wavelength of 350 nm and emission wavelength of 420 nm and slit width of 5.0 nm. Fluorescence measurements were taken in triplicate and averaged.

solids. The crude peptides were confirmed by ESI mass spectra and were used for biological testing without any further purification. Bacterial Strains and Growth Media. Ea1501 was obtained from the International collection of Microorganisms for Plants (Landcare Research, New Zealand). E. coli DH5α, P. aeruginosa, and S. aureus were obtained from the School of Biological Sciences, microbial culture collection. Str4Ea and Psa16027 were a gift by Dr. Vanneste, New Zealand Institute for Plant and Food Research. The strains were stored in 50% glycerol at −20 °C and −80 °C. For routine use, bacteria were plated on nutrient agar plates containing proteose peptone no. 3 (0.5%), yeast extract (0.3%), NaCl (0.5%), and agar (2.5%) dissolved in 500 mL of sterilized Milli-Q water and stored in the fridge. Single colonies were frequently replated every 2 weeks. Yeast extract, select agar, and Luria broth were obtained from Invitrogen. Proteosepeptone No.3 was obtained from BD Biosciences. All media for bacterial growth and glassware were autoclaved at 120 °C for 1 h using a Tomy SX 500E high pressure steam sterilizer. Determination of Minimal Inhibitory Concentration (MIC). Luria broth base (1.25%) was dissolved in 100 mL of sterilized Milli-Q water and autoclaved at 120 °C for 1 h and cooled to room temperature. A single colony of the bacterial strain was transferred to 20 mL of Luria broth and grown at either 28 °C (Ea1501, Str4Ea, Psa16207) or 37 °C (E. coli and S. aureus) overnight. The optical density of the overnight culture was measured and adjusted to be 0.6 (600 nm) by diluting with Luria broth. Peptides and the antibiotic controls were dissolved in Luria broth with 0.5% acetic acid and a dilution series made. The assay was performed by adding 50 μL each of the peptide solutions at various concentrations and 50 μL of the diluted bacterial culture to the different wells of a 96-well microtiter plate. Three replicates of each peptide concentration were tested against each bacterial strain. MIC was defined as the lowest peptide concentration required to inhibit the growth of bacteria after 24 h of incubation determined by both visual inspection and absorbance at 600 nm. Optical density measurements for the MIC assay were conducted using an EnSpire Multimode plate reader. Kinetics of Killing. An overnight bacterial culture was diluted to mid logarithmic phase with Luria broth to yield 105−108 CFU/mL. The diluted bacterial solution (100 μL) was added to 10 mL of the antibacterial peptide solution sterile LB at 1×, 2×, 4×, and 20× MIC. The solutions were incubated at either 28 °C (Ea1501, Str4Ea and Psa16027) or 37 °C (E. coli, P. aeruginosa and S. aureus) with shaking (100 rpm) and 100 μL was pipetted onto Luria Broth agar plates at 0, 0.5, 1, 3, 6, 9, and 24 h by doing appropriate serial dilutions in 0.9% sterilized saline to result in approximately 20−300 colonies. After 24 h of incubation, the surviving colonies were counted. The experiment was done in duplicate. The effect observed was defined as bactericidal if a ≥ 2-log10 CFU/mL decrease compared to the positive control (no antibiotic) was observed.18 Inhibition of Psa16027 Biofilm Formation: Semiquantitative Estimation and Visualization of Crystal Violet Stained Biofilms. For semiquantitative estimation, biofilms of Psa16027 were prepared in 96-well plates using an overnight culture of Psa16027 (OD600: 0.3) grown at 28 °C in Luria broth medium with sufficient number of the peptide (17) test samples, positive and negative controls. The assay was performed by adding 50 μL of 17 in sterile LB at various concentrations (1−100 μM) and 50 μL of the diluted bacterial culture to the different wells of the 96-well microtiter plate. Positive control comprised of 50 μL of the diluted bacterial culture and 50 μL of LB medium. Negative control comprised of sterile LB medium (100 μL). Three replicates of each peptide concentration were tested. The plates were incubated for 48 h at 28 °C at 50 rpm. At appropriate time intervals, supernatant from each well was discarded and the wells stained with 100 μL of 1% crystal violet for 5 min, following standard protocols.43 The stain was removed carefully, and the wells washed twice with 100 μL of sterile water. Crystal violet stained biofilms at the bottom of each well were solubilized with 96% ethanol and the absorbance of this ethanol solution measured at 560 nm for a semiquantitative estimation of biofilm biomass. For imaging purposes, biofilms of Psa16027 were prepared in 12well plates as described above. Lipopoeptide 17 (50 μL) at 1, 5, and 50 636

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Journal of Medicinal Chemistry Propidium Iodide (PI) Uptake Assay.23,44,45 P. aeruginosa and S. aureus were grown overnight in LB at 37 °C. The cells were then reincubated in fresh LB for 3 h to OD600 of 0.35. The bacterial cells were harvested by centrifugation at 4600 rpm for 10 min and washed with 10 mM phosphate buffer (pH 7) thrice. The bacterial cells were resuspended in phosphate buffer to a final concentration of 108 CFU/ mL. The cell suspension (50 μL) was incubated with 50 μL of peptide solutions at various concentrations (0.5−100 μM) in phosphate buffer for 60 min at 37 °C. Then 5 μL of propodium iodide (PI) at 1 μM was added to the cell suspension and fluorescence was measured using a multimode microplate reader at excitation and emission wavelengths of 520 and 620 nm, respectively. Hemolytic Assay. The hemolytic activity of the lipopeptides was tested by determining the extent of hemoglobin release from erythrocyte suspensions of fresh mouse blood cells (2% vol/vol). Freshly collected mouse blood cells were centrifuged at 1000g for 5 min to remove the buffy coat. The blood cells were washed thrice in Tris buffer (10 mM Tris, 150 mM NaCl, pH 7.4) and resuspended in 2% (v/v) of the Tris buffer. The peptides were dissolved in Tris buffer at concentrations ranging from 1 mM to 1 μM. The peptide solution (100 μL) was added to resuspended blood cells (100 μL) in 96-well plates and the plates incubated for 1 h at 37 °C without agitation. The buffer solution and 0.1% Triton X-100 were used as the negative and positive controls, respectively. Each sample and controls were tested in triplicate. The plates were centrifuged at 3500g for 10 min. Supernatant from each sample (100 μL) was transferred to new 96well plates and the absorbance at 540 nm measured. Percentage hemolysis at each peptide concentration was calculated from the following equation, where Aexp is the experimental A540 measurement, ATris is the negative control where only Tris buffer was added to RBC, and A100%, is the positive control where 0.1% Triton X-100 was used to cause lysis of 100% RBC present.

further resonance assignments and analysis. Spectral width was set to 9600 Hz (12 ppm). Two K time domain points were used in direct dimension (t2) with 400 points in the indirect dimension (t1). The data were zero filled into 1K points in t1 dimension to improve resolution. TOCSY and ROESY mixing times were 100 and 250 ms, respectively. All data processing was done offline using TOPSPIN (version 2.1). Shifted sine bell window functions were used for processing 2D data. Scanning Electron Microscopy. E. coli, P. aeruginosa, S. aureus, and Psa 16207 were grown to an optical density of 0.3 in Luria broth with glass slides (10 mm) for 24 h. Excess bacteria were removed by washing with 10 mM sodium phosphate buffer (pH 7.4). The glass slides were treated for 20 min, with the peptide dissolved in Luria broth at twice the upper limit of its MIC. The excess peptide was removed by washing with 10 mM PBS. The glass slides were fixed with 4% glutaraldehyde in 10 mM phosphate buffer (pH 7.4) for 1 h. The slides were washed with 10 mM sodium phosphate buffer and dehydrated with graded ethanol series (25−100%) prior to drying at 60 °C for 10 min. The dried slides were platinum coated for 2 min at 20 mA conditions before viewing under high vacuum with a FEI Quanta 200 F ESEM microscope at 10 kV. Molecular Modeling. A model of the peptide was generated on computer using Accelyrs discovery studio 3.5. A restrained energy minimization was carried out in vacuum using NMR derived distance and torsion constraints as described in detail below. The linear model peptide was built using Hyperchem or chemdraw and converted to a *.mol or *.pdb file. From NMR data, distance and dihedral constraints were obtained. The distance constraints were from NOE data which were broadly classified into three categories as strong, medium, and weak, and the corresponding upper limit set was 2.5, 3.5, and 5.0 Å, respectively, while the lower limit in all cases were fixed at 1.9 Å. A strong NOE across the Dab(6) CγH2 geminal protons resonating at 2.63 and 2.48 ppm was considered as reference. A loose ϕ dihedral constraint of 0° to −180° was used for all L-residues as their 3JNHCαH values were around 7 Hz. The NMR derived distance and dihedral constraints were applied. The structure was refined by energy minimization using CHARMm in Discovery Studio 3.5, which provides powerful mechanics and dynamics protocols for studying the energetic and motion of molecules, from small ligands to multicomponent physiological complexes. Accelrys CHARMm force field was used throughout the simulation. Approximately 2000 cycles of steepest descent gradient was applied during minimization to clean the geometry of the peptide followed by 500−1000 steps of conjugate gradient. The refined model was validated with Ramachandran plot.

%haemolysis = (Aexp − A Tris)/(A100% − A Tris)× 100 Circular Dichroism. Circular dichroism (CD) spectra were recorded on an Applied Photophysics δ-Star 180 spectrometer, at 20 °C in a 0.1 cm path-length cuvette, from 190 to 250 at 0.5 nm intervals with a 5 s response time. Six scans per sample were averaged. Data are expressed as mean residue ellipticities [θ] in deg × cm2 dmol−1. Peptide stock solutions were prepared in 10 mM sodium phosphate buffer (pH 7.0), methanol, trifluoroethanol (TFE), or 2 mM DMPCPDA vesicles and diluted to 500 μM concentration under inert conditions using the respective solvents. The vesicles were prepared as described below. Dimyristoylphosphatidylcholine (DMPC; Avanti Polar Lipids, Inc. Alabama, USA) and 10,12-tricosadiynoic acid (TRCDA; Alfa Aesar Heysham, England) were dissolved in a 4:6 ratio in DCM and dried together under nitrogen to yield a thin white film. This film was dissolved in Milli-Q water to a total lipid concentration of 2 mM. This solution was probe sonicated for 10 min at 80−90 °C and cooled to room temperature before being kept overnight at 4 °C. Prior to polymerization, vesicles were warmed to room temperature and polymerized by irradiation with UV light (254 nm) until a deepblue color was formed. Percentage helicity was calculated as follows: αhelix (%) = ([θ]222 − [θ]2220)/([θ]222100) where [θ]222 is the experimentally observed absolute mean residue ellipticity at 222 nm. Values for [θ]2220 and [θ]222100, corresponding to 0 and 100% helix content at 222 nm, were estimated to be −3000 and −36000 (deg × cm2 dmol−1), respectively.46,47 NMR Experiments. Data Acquisition. All 1D and 2D NMR experiments of 17 were carried out on either Bruker 800 or 700 MHz spectrometer attached with a cryoprobe, at the NMR research center, Indian Institute of Science, Bangalore. All 500 and 600 MHz NMR spectra were recorded on Bruker NMR spectrometer at the University of Auckland, New Zealand. A series of 1D spectra of 17 were recorded at five different temperatures between 278 and 318 at 10 K intervals. From these spectra, amide proton temperature coefficients (dδ/dT) were calculated. At 278 K, in addition to dispersion being good, there was also sharpening of side chain amide resonances due to slow exchange rates. Hence all 2D spectra were recorded at 278 K for

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ASSOCIATED CONTENT

S Supporting Information *

Analytical HPLC traces, ESI-MS and 1H NMR spectra of all peptides, TOCSY and NOESY spectra of 17 in CD3OH and water−TFE mixtures, plots of amide proton chemical shifts against temperature in these solvents and relevant biofilm images. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*For V.S.: phone, + 64 9 923 3387; E-mail, v.sarojini@ auckland.ac.nz. *For S.R.: phone, +91 80 22933301; E-mail, [email protected]. in. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS S.R. acknowledges financial support from the Department of Science and Technology, Government of India. We also thank 637

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Journal of Medicinal Chemistry

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V. Viswanath and M. Schmitz for help with NMR data collection and Z. Wu with haemolytic assay.

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ABBREVIATIONS USED AMPs, antimicrobial peptides; CFU, colony forming units; Dab, α,γ-diaminobutyric acid; DIPEA, N,N-diisopropylethylamine; DMF, N,N-dimethylformamide; DMPC, Dimyristoylphosphatidylcholine; Ea1501, Erwinia amylovora 1501; E. coli, Escherichia coli; Fmoc, fluorenylmethyloxycarbonyl; HOBt, 1hydroxybenzotriazole hydrate; LB, luria broth; MIC, minimal inhibitory concentration; Mtt, 4-methyltrityl; PBS, phosphate buffered saline; P. aeruginosa, Pseudomonas aeruginosa; PDA, polydiacetylene; PI, propidium iodide; PMB, polymyxin B; RPHPLC, reverse-phase high pressure liquid chromatography; NPN, 1-N-phenylnaphthylamine; Psa, Pseudomonas syringe pv. actinidiae; Str4Ea, streptomycin resistant Erwinia amylovora; S. aureus, Staphylococcus aureus; TBTU, O-(benzotriazol-1-yl)N,N,N′,N′ tetramethyluronium tetrafluoroborate

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