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In vitro antibacterial and antimalarial activity of dehydrophenylalanine-containing undecapeptides alone and in combination with drugs
International Journal of Antimicrobial Agents 39 (2012) 146–152
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In vitro antibacterial and antimalarial activity of dehydrophenylalanine-containing undecapeptides alone and in combination with drugs Sarika Pathak Sharma a , Jyotsna Sharma a,1 , Shamsher Singh Kanwar b , Virander Singh Chauhan a,∗ a b
International Centre for Genetic Engineering and Biotechnology, New Delhi 110067, India Department of Biotechnology, Himachal Pradesh University, Shimla 171005, India
a r t i c l e
i n f o
Article history: Received 11 August 2011 Accepted 6 October 2011 Keywords: Antimicrobial peptides De novo design Dehydrophenylalanine Antiplasmodial activity
a b s t r a c t A set of three cationic undecapeptides, analogous to the previously reported peptide VS2 (KWFWKFVKFVK), was created by alanine substitution in order to probe the effect of hydrophobicity on peptide activity. The activities of these peptides were determined against Escherichia coli, Staphylococcus aureus and the malaria parasite Plasmodium falciparum. VA1, the closest analogue of VS2, showed five-fold augmented activity [minimum inhibitory concentration (MIC) = 10 M] against the Gram-positive bacterium S. aureus. The designed analogues were non-haemolytic and non-cytotoxic at their MICs and clinically relevant concentrations. By alanine substitution, it was also possible to probe the critical role of tryptophan residues in determining peptide potency. Circular dichroism studies of the peptides in a membrane-mimetic system showed a correlation between peptide helicity and antimicrobial activity. The peptides were also tested in combination with sublethal concentrations of antibiotic drugs (rifampicin and kanamycin) and the antimalarial drug chloroquine. In combination with these drugs, the effect of the peptides was synergistic or additive. These results provide insight into basic design principles for generating new clinically relevant lead peptides. It also provides an alternative strategy where a peptide and a non-peptide drug can be used in combination to battle increasingly drug-resistant microbes. © 2011 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.
1. Introduction The widespread use of antibiotics in recent years has led to the development of microbial strains that are resistant to almost all existing antibiotics [1]. This generates an urgent need for either new antibiotics or an alternative strategy such as combination therapy, where a combination of two or more drugs are used for the treatment of infection [2]. In the development of newer antibiotics, host defence peptides or cationic antimicrobial peptides (AMPs) have emerged as potential therapeutic agents, primarily because the likelihood of development of resistance to them is very low [3]. In addition, AMPs possess a broad spectrum of antimicrobial activity against Gram-positive and Gram-negative bacteria, fungi and protozoa [4]. Some of them are also known to have antiviral and anticancer activities [5,6]. Despite being suitable candidates for tackling microbial resistance, clinical development of naturally occurring AMPs has been hampered because of their poor
∗ Corresponding author. Tel.: +91 11 2674 1358; fax: +91 11 2616 2316. E-mail address: [email protected] (V.S. Chauhan). 1 Present address: FB Biology, Philipps-Universität, 35032 Marburg, Germany.
bioavailability, large size and mammalian cell cytotoxicity [7]. Synthetic or designer AMPs have recently become a focus for drug design owing to high-throughput studies that generated multiple synthetic peptides active against a broad range of pathogens [8,9]. In our previous work, we have reported the de novo design of a series of AMPs with activity towards the Gram-negative bacterium Escherichia coli and the Gram-positive bacterium Staphylococcus aureus [10]. These peptides incorporated a non-natural amino acid, dehydrophenylalanine, which is the dehydro analogue of the amino acid phenylalanine and is found in the antibiotic peptides of fungal origin such as tentoxin [11]. The presence of these residues in peptides has been shown to impart relative protease stability to peptides [12]. In this study, the sequence of a previously designed undecapeptide (VS2) was optimised in terms of high antibacterial activity and low eukaryotic cell toxicity. Three undecapeptides analogous to VS2 were generated and their biological activities were studied. Here we describe the in vitro activities of these peptides against E. coli and S. aureus (meticillin-resistant). We also show the increased effectiveness of these peptides at low concentrations when they are used in combination with the antibiotics kanamycin and rifampicin. Since these peptides were expected to have no or negligible haemolysis, their activity against the malaria
0924-8579/$ – see front matter © 2011 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved. doi:10.1016/j.ijantimicag.2011.10.008
S.P. Sharma et al. / International Journal of Antimicrobial Agents 39 (2012) 146–152
parasite Plasmodium falciparum was also determined, both independently and in combination with a sublethal concentration of the conventional antimalarial drug chloroquine (CQ). 2. Materials and methods 2.1. Materials Bacterial strains E. coli ML35p and S. aureus ATCC 700699 were kind gifts from Dr Dinkar Sahal [International Center for Genetic Engineering and Biotechnology (ICGEB), New Delhi, India]. Amino acid derivatives and resin for peptide synthesis were obtained from Novabiochem (Merck, Darmstadt, Germany). Diisopropylcarbodiimide (DIPCDI), piperidine, dimethylformamide (DMF), dichloromethane, hydroxybenzotriazole (HOBt), isobutyl chloroformate, trifluoroacetic acid (TFA), triisopropylsilane, dl-threo--phenylserine, sodium hydroxide, citric acid, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), dimethyl sulphoxide (DMSO), bovine serum albumin (BSA) and N-methylmorpholine were from Sigma-Aldrich (St Louis, MO). RPMI 1640 and fetal bovine serum (FBS) were from Invitrogen (Carlsbad, CA). Sodium chloride, acetic anhydride and tetrahydrofuran were from Qualigens (Mumbai, India). Ethyl acetate, diethyl ether, sodium acetate and sodium sulphate were from Merck (Mumbai, India). Silica gel thin-layer chromatography plates (60F-254) were from Merck. Acetic acid was from SD Fine-Chem Ltd. (Mumbai, India). Acetonitrile was from Burdick and Jackson (Muskegon, MI). 2.2. Peptide synthesis Peptides were synthesised as C-terminal amides using standard Fmoc chemistry on rink amide MBHA (4-methylbenzhydrylamine hydrochloride salt) resin in the manual mode, with DIPCDI and HOBt as coupling agents. Peptides were acetylated at the Nterminus and amidated at the C-terminus. Both the coupling of amino acids and the Fmoc deprotection were monitored by the Kaiser test [13]. The amino acid dehydrophenylalanine (Phe) was introduced into peptides as an Fmoc-X-Phe azalactone [where X is Lys (Boc), Trp (Boc) or Ala] dipeptide block, which was allowed to couple overnight in DMF [10,12]. 2.3. Peptide purification and mass spectrometry Crude peptides were purified by reverse-phase highperformance liquid chromatography using a water–acetonitrile gradient on a C18 Phenomenex column (2 × 15 cm, 15 mm column length, flow rate 5 mL/min; Shimadzu, Kyoto, Japan); gradient of 5% to 65% acetonitrile and 0.1% TFA for 70 min, with detection at 214 nm and 280 nm. The identity of the highly purified (95%) peptides was confirmed by electrospray ionisation mass spectrometry performed at the ICGEB. Concentrations of the peptide stocks were determined by measurement of the absorbance at 280 nm [ 280, 19 000 M−1 cm−1 for ␣,-didehydrophenylalanine (Phe) and 5050 M−1 cm−1 for tryptophan]. 2.4. Antibiotic susceptibility testing Escherichia coli and S. aureus were grown overnight and were diluted in Mueller–Hinton broth (DifcoTM ; Becton Dickinson, Sparks, MD) to a cell density of 105 colony-forming units/mL. Then, 100 L of this culture was aliquoted into the wells of a 96well flat-bottomed microtitre plate (Costar; Corning, Acton, MA) and 11 L of 10X stock of each peptide concentration (in 0.2% BSA and 0.01% acetic acid) was added. Three wells were used for each concentration. The plate was incubated at 37 ◦ C in a
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rotary shaker incubator (Kühner, Basel, Switzerland) set at 180 rpm. After 18 h of incubation, the optical density at 600 nm (OD600 ) was measured using a microtitre plate reader (VersaMaxTM tunable microplate reader; Molecular Devices, Sunnyvale, CA). The minimum inhibitory concentration (MIC) was defined as the lowest concentration of drug that inhibited measurable growth of an organism following overnight incubation. Each set of experiments was repeated at least twice. The MICs of peptides in combination with the drugs kanamycin and rifampicin (Sigma-Aldrich, St Louis, MO) at the concentration that inhibits 50% of microorganism growth (MIC50 ) were also determined by the above protocol. Peptides in combination with 2 M kanamycin (MIC50 ) were used against E. coli, whereas peptides combined with 500 nM rifampicin (MIC50 ) were used against S. aureus.
2.5. Haemolytic activity testing Human blood in 10% citrate–phosphate–dextrose was obtained from the Rotary Blood Bank (New Delhi, India). Red blood cells (RBCs) were harvested by spinning (1000 × g, 5 min, room temperature). RBCs were washed three to five times with phosphate-buffered saline (PBS). The packed cell volume obtained was used to make a 0.8% (v/v) suspension in PBS. Then, 100 L of this RBC suspension was transferred to each well of a 96-well microtitre plate and was mixed with 100 L of peptide solution at twice the desired concentration. The microtitre plate was incubated (37 ◦ C, 60 min) and centrifuged (1000 × g, 5 min, room temperature). The supernatant (100 L) was transferred to new wells and the OD414 was measured with a microtitre plate reader to monitor RBC lysis. Cells incubated with PBS alone acted as the negative control, and RBCs lysed using 0.1% Triton X-100 were used to measure 100% lysis.
2.6. Mammalian cell cytotoxicity Cytotoxicity of the antibiotic peptides was determined using the MTT assay against HeLa and fibroblast L929 cells. Briefly, cells (104 cells/well) were cultured at 37 ◦ C overnight in RPMI 1640 containing 10% FBS in 96-well microtitre plates. The next day, peptides (prepared in RPMI 1640) at concentrations of 1X and 5X MIC values were added to the cells and were incubated for 18 h at 37 ◦ C. DMSO (10%) was taken as the positive control and untreated cells served as the negative control. Then, 20 L of MTT solution (5 mg/mL) in PBS was added and the cells were incubated at 37 ◦ C for 3–4 h. Supernatant (120 L) was removed, DMSO (100 L) was added and the resulting suspension was mixed to dissolve the formazan crystals formed by MTT reduction. The ratio of OD570 for treated cells to OD570 for untreated cells was used to calculate percent viability.
2.7. Circular dichroism (CD) spectrometry CD studies were performed at 25 M peptide concentrations in 20 mM sodium dodecyl sulphate (SDS) (USB Corp., Cleveland, OH) in 10 mM phosphate buffer (pH 7.4). All spectra were recorded on a JASCO J-810 polarimeter (Jasco, Tokyo, Japan) equipped with a Peltier-type thermostat and purged continuously with dry N2 gas at 10 L/min during data acquisition. Data were collected in a quartz cuvette with path length 1 cm, between 190 nm and 340 nm at a scan speed of 50 nm/min and response time of 8 s. The average of five scans was used for analysis of the spectrum. The CD spectra of peptides were baseline-corrected by subtracting the CD spectra of SDS in buffer.
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2.8. Antiplasmodial activity of peptides 2.8.1. In vitro cultivation of Plasmodium falciparum The CQ-sensitive strain 3D7 of P. falciparum was used in in vitro blood-stage culture to test the antimalarial efficacy of the peptides VA1, VA2 and VA3 alone and in combination with 40 nM CQ. Plasmodium falciparum 3D7 cultures were maintained in fresh O-positive human erythrocytes suspended at 4% haematocrit in RPMI 1640 (Sigma) containing 0.2% sodium bicarbonate, 0.5% AlbuMAX (Sigma-Aldrich), 45 g/L hypoxanthine (Sigma-Aldrich) and 50 g/L gentamicin (Sigma-Aldrich) and incubated at 37 ◦ C under a gas mixture of 5% O2 , 5% CO2 and 90% N2 . 2.8.2. Peptide and drug dilutions Peptide and CQ stocks were prepared in Milli-Q water (Millipore, Billerica, MA) and were serially diluted with Phenol red-free RPMI 1640 to achieve the required concentrations. When used in combination, a fixed concentration (40 nM) of CQ was added to each serially diluted peptide concentration. CQ and test peptides were then placed in 96-well flat-bottom tissue culture-grade plates. 2.8.3. Assay for antiplasmodial activity and haemolysis of parasitised red blood cells A SYBR Green® -based fluorescence assay was set-up as previously described [14]. Sorbitol-synchronised parasites were incubated under normal culture conditions at 2% haematocrit and 1% parasitaemia in the absence or presence of increasing concentrations of peptide alone or in combination with CQ. CQ was also used as a positive control, whilst 0.2% Triton X-100 was used as the negative control. After 48 h of incubation, plates were spun at 2500 × g for 10 min at 25 ◦ C. Then, 100 L of the supernatant was taken and transferred to another plate for determination of haemolysis at 414 nm. In brief, 100 L of SYBR Green I solution (Invitrogen) (0.2 L of 10,000 X SYBR Green I/mL) in lysis buffer [20 mM Tris (pH 7.5), 5 mM ethylene diamine tetra-acetic acid, 0.008% (w/v) saponin and 0.08% (v/v) Triton X-100] was added to each well, mixed gently and incubated in the dark at 37 ◦ C for 1 h. Fluorescence was measured with a Victor fluorescence multiwell plate reader (Perkin-Elmer, Waltham, MA) with excitation and emission wavelength bands centred at 485 nm and 530 nm, respectively. The fluorescence counts were plotted against the drug concentration, and the 50% inhibitory concentration (IC50 ) was determined by analysis of dose–response curves. 3. Results 3.1. Peptide analogue design In this study, three new AMPs analogous to our previously reported lead broad-spectrum undecapeptide VS2 were designed and synthesised. The analogues were identical to VS2 in length and net positive charge (four lysine residues); however, they had alanine substitutions at one or two places on the hydrophobic face. Similar to VS2, these analogues also harboured three F residues on the hydrophobic face (Table 1). Fig. 1 shows a helical wheel representation of the basic peptide sequence and positions of Ala substitutions. VA1 had a single Ala substitution for Val at position 10. In VA2, two Ala were substituted, one for a Trp at position 2 and another for Val at position 10. In VA3, both the Val residues (positions 7 and 10) were replaced by Ala. 3.2. Antibacterial activity of peptides The peptide analogues VA1 and VA3 retained their antibacterial activity in the low micromolar range both against Gram-negative E. coli and Gram-positive S. aureus (Table 2). VA1 showed a MIC
Fig. 1. Basic template of the peptide analogues. (A) A 310 helical wheel representation of the peptides showing a hypothetical segregation of the hydrophobic and hydrophilic faces. White box, Ala substitution at this position was well tolerated; grey box, there was a three-fold decrease in the activity against Escherichia coli, however activity against Staphylococcus aureus was moderately improved; black box, Ala substitution at this position drastically reduced the potency of the peptide. (B) Chemical structure of the amino acid ␣,-dehydrophenylalanine (F).
of 4 M against E. coli and 10 M against S. aureus, whereas VA3 showed a MIC of 15 M for E. coli and 35 M for S. aureus. Compared with VS2, no gain in the activity of VA1 and VA3 against E. coli was observed. However, VA1 showed a five-fold lower MIC against S. aureus and VA3 also showed a reduced MIC value for S. aureus compared with VS2. VA2 was found to be only moderately active against E. coli (MIC = 75 M) and had no activity against S. aureus up to 125 M. 3.3. Antibiotic activity of peptides in combination with kanamycin and rifampicin The antibacterial activity of peptides in combination with known antibiotic drugs against S. aureus and E. coli was also tested and it was demonstrated that these peptides acted synergistically with the antibiotic drugs (Fig. 2). 3.3.1. Activity against Escherichia coli in combination with a sublethal concentration of kanamycin Since kanamycin has a MIC of ca. 4 M against E. coli ML35p, 2 M kanamycin (MIC50 ) was used in combination with different concentrations of peptides. The results show that VA1 and VA3 showed only a moderate decrease in MIC (additive effect) in combination with drug, whilst VA2 showed a very good synergy since its MIC value decreased from 75 M to 30 M. Killing curves of peptides alone and in combination with kanamycin show that the growth of the bacteria was inhibited at very early stages when the peptide was used in combination (Fig. 2A). 3.3.2. Activity against Staphylococcus aureus in combination with a sublethal concentration of rifampicin Rifampicin, a non-peptide antibiotic, has a MIC of 1 M against S. aureus [10]. An additive effect was also observed against S. aureus when VA1 and VA3 were used in combination with 500 nM rifampicin (MIC50 ). The MIC of VA1 decreased from 10 M to 7.5 M and that of VA3 decreased from 35 M to 30 M. However, VA2 again showed very good synergy with the drug. In
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Table 1 Amino acid sequences of peptides. Residues
Charge
F
Ala
MW (Da)
Ac-K-W-F-W-K-F-V-K-F-V-K-NH2
11
+4
3
0
1575
Ac-K-W-F-W-K-F-V-K-F-A-K-NH2 Ac-K-A-F-W-K-F-V-K-F-A-K-NH2 Ac-K-W-F-W-K-F-A-K-F-A-K-NH2
11 11 11
+4 +4 +4
3 3 3
1 2 2
1549 1433 1521
Peptide sequence VS2 Alanine analogues VA1 VA2 VA3
MW, molecular weight.
Table 2 Minimum inhibitory concentrations (MICs) of peptides against the Gram-positive bacterium Staphylococcus aureus and the Gram-negative bacterium Escherichia coli, and 50% inhibitory concentrations (IC50 ) of peptides against the malaria parasite Plasmodium falciparum alone and in combination with 40 nM chloroquine (CQ). Peptide/Drug
MIC (M)
IC50 against P. falciparum (M)
E. coli ML35p VS2 VA1 VA2 VA3 Rifampicin Kanamycin
5 4 75 15 5 4
50 10 >125 35 1 7.5
Peptide alone
In combination with CQa
N/D 10 >40 15 – –
N/D 2.5 10 7.5 – –
40 nM is the IC50 value of CQ for P. falciparum 3D7 strain.
0.8
0.8
E. coli (ML35p)
0.7 0.6 OD 600
S. aureus (ATCC 700699)
0.7 0.6
VA1
0.5 VA1+Kanamycin (2μM)
0.4 0.3
OD 600
a
S. aureus ATCC 700699
VA1
0.5
VA1+ Rifampicin (500 nM)
0.4 0.3
0.2
0.2
0.1
0.1
0
0 0
1
2
3
4
5
10
0
1
0.8
0.5
15
20
S. aureus (ATCC 700699)
0.6
VA2
VA2+Kanamycin (2μM)
0.5
VA2+Rifampicin (500nM)
0.4 0.3
0.4 0.3
0.2
0.2
0.1
0.1 0
0 0
5
10
20
30
40
0
50
5
10
Peptide conc (μM) E. coli (ML35p)
0.8
VA3
0.7
0.6
VA3+ Kanamycin (2μM)
0.6
25 50 75 Peptide conc (μM)
0.4 0.3
VA3+Rifampicin (500nM)
0.4 0.3
0.2
0.2
0.1
0.1
0
125
VA3
0.5
0.5
100
S. aureus (ATCC 600799)
0.8
0.7
OD 600
OD 600
12.5
VA2 OD 600
OD 600
0.8 0.7
0.6
10
Peptide conc (μM)
E. coli (ML35p)
0.7
7.5
5
Peptide conc (μM)
0 0
0.5
1
2.5
5
Peptide conc (μM)
(A)
10
15
0
1
5
7.5
10 12.5 15
20
25
30
35
Peptide conc (μM)
(B)
Fig. 2. Antibacterial activities of peptides alone and in combination with antibacterial drugs at a concentration that inhibits 50% of microorganism growth (MIC50 ). (A) Growth inhibition curve of Escherichia coli ML35p treated with peptide alone and in combination with 2 M kanamycin (MIC50 ). (B) Growth inhibition curve of Staphylococcus aureus ATCC 700699 treated with peptide alone and in combination of 500 nM rifampicin (MIC50 ). OD600 , optical density at 600 nm.
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(A)
90
VA1
VA1 2.25
VA2
80
VA3
70
1.75
60
θx105 (deg cm2 mol-1
% Hemolysis
VA2
VA3
50 40 30 20
1.25
0.75
0.25 10 0
-0.25 1μM
5μM
10μM
25μM
50μM
100μM
190
210
230
250 270 Wavelength (nm)
290
310
330
Peptide conc -0.75
(B)
HeLa cell cytotoxicity
120
VA1 VA2
% Viability
100
Fig. 4. Circular dichroism spectra of peptides at 25 M in 20 mM sodium dodecyl sulphate in phosphate buffer (pH 7.4).
VA3
80
haemolysis, respectively, and VA3 showed 15% and 20% haemolysis. VA2 was least haemolytic at 100 M and showed only 6% haemolysis (Fig. 3A). Similarly, in the case of HeLa and fibroblast L929 cells, no significant cytotoxicity was observed for any peptide at MIC values (Fig. 3B and C). However, at 50 M concentration they were 10–20% cytotoxic and at 100 M they were 30–50% cytotoxic. Only VA1 showed 40–50% toxicity at 25 M and was toxic above that concentration.
60
40
20
0 5μM
10μM
25μM
50μM
100μM
3.5. Secondary structure of peptides
Peptide conc
(C)
Fibroblast L929 cell cytotoxicity 120
VA1 VA2 VA3
100
% Viability
80
60
40
CD studies show that the peptides acquired helical structures in a membrane-mimetic environment of 20 mM SDS (Fig. 4). All of the peptides exhibited an excitonic couplet at 267 nm (+) and 298 nm (−), which is the signature for the formation of a right-handed 310 helix [15] (Fig. 4). VA3 was found to be most helical (measured in terms of band intensity at 267 nm), followed by VA1 and VA2 (least helical). Correlation was also observed between helicity and activity of the peptides. The least helical peptide VA2 was also poor in potency. Increased helicity resulted in increased potency of the peptide in VA1. However, further increase in helicity resulted in lower potency of VA3 compared with VA1.
20
0 5μM
10μM
25μM
50μM
100μM
Peptide conc Fig. 3. (A) Percent haemolysis of human red blood cells after 1 h of incubation with peptides. Haemolysis by 0.2% Triton X-100 was taken as 100%. (B and C) Percent viability of HeLa cells (B) and fibroblast L929 cells (C) after 24 h of incubation with peptides in an MTT-based cell viability assay. Dimethyl sulphoxide (DMSO) (10%) was taken as control for 100% cytotoxicity.
combination, its MIC was reduced to 75 M against S. aureus whereas alone it showed no activity up to 125 M (Fig. 2B). 3.4. Cell selectivity: haemolysis and cytotoxicity to HeLa and L929 cells None of the peptides showed noteworthy haemolysis or cytotoxicity to HeLa and L929 fibroblast cells at their respective MICs (Fig. 3). However, at higher concentrations (≥50 M), VA1 and VA3 showed some degree of haemolysis and cytotoxicity. At 50 M and 100 M concentrations, VA1 showed 30% and 75%
3.6. Antiplasmodial activity of peptides alone and in combination with chloroquine Since many natural and synthetic AMPs have been shown to have antimalarial activity, the antiplasmodial activity of the designed peptides was also tested, independently and in combination with the antimalarial drug CQ using a standard SYBR Green assay (Table 2). SYBR Green is a DNA-binding cell-permeant fluorescent dye with excitation/emission at 485/535 nm [16]. The results obtained with this assay showed IC50 values of 10 M, >40 M and 15 M for VA1, VA2 and VA3, respectively. It was also shown that in combination with 40 nM CQ the IC50 values of the peptides decreased to 2.5 M, 10 M and 7.5 M for VA1, VA2 and VA3, respectively. Furthermore, haemolysis of parasitised RBCs after 48 h of incubation with peptide in combination with CQ was determined in the same experimental assay. As shown in Fig. 5, no significant haemolysis was observed even after 48 h. These results suggest that the peptides showed synergy with CQ, and such combinations can be extremely useful against the development of alarmingly increasing CQ-resistant strains of P. falciparum.
% Haemolysis of parasitised RBCs
S.P. Sharma et al. / International Journal of Antimicrobial Agents 39 (2012) 146–152 20
VA1 VA2 VA3
15
10
5
0
Peptide conc+Cq conc Fig. 5. Percent haemolysis of parasitised red blood cells incubated with peptides and 40 nM chloroquine for 48 h. Haemolysis by 0.2% Triton X-100 was taken as 100%.
4. Discussion We have previously reported rational-based design of short AMPs that act by increasing bacterial membrane permeability [10]. However, despite being potent against bacteria, some of them were non-specific in their activity and also showed RBC haemolysis and mammalian cell cytotoxicity. In that work, we had optimised the sequence of peptide VS2 in terms of potency, non-cytotoxicity and faster killing kinetics. However, we also reported that at high concentrations (well above the MIC for E. coli) the peptide caused haemolysis and cytotoxicity and required further improvement in the design in order to be a suitable drug candidate. Literature shows that the lack of cell selectivity is related to high hydrophobicity and high amphipathicity of the peptides [17]. Nevertheless, a certain degree of amphipathicity and hydrophobicity is crucial for interaction with negatively charged microbial membranes [18]. In an effort to improve peptide specificity, we tried to alter the peptide hydrophobicity and amphipathicity by replacing more hydrophobic and bulky residues (Val and Trp, respectively) present on the hydrophobic face of the peptides with a moderately hydrophobic residue Ala. We tried to reduce the overall hydrophobicity of the peptide using the following designs: replacement of single Val with Ala (VA1); replacement of one Trp and one Val each with Ala (VA2); and replacement of two Val with two Ala (VA3). This study on Ala analogues showed that reduced hydrophobicity indeed resulted in altered cell selectivity of peptides. In VA1, replacement of a single Val by Ala at the hydrophilic face only slightly lowered the hydrophobicity of the peptide. This substitution did not alter the activity of the peptide against E. coli and eukaryotic cells. However, it enhanced its potency five-fold against Gram-positive S. aureus. The augmented activity against S. aureus can be explained by the difference in the membrane compositions of Gram-negative and Gram-positive bacteria and the eukaryotic membrane. The eukaryotic membrane is zwitterionic, whereas the membrane of Gram-negative bacteria is negatively charged and partially zwitterionic and that of Gram-positive bacteria is completely negatively charged [19]. Literature shows that increased hydrophobicity of peptides increases their interaction with zwitterionic membranes (eukaryotic and Gram-negative membranes) but not with the negatively charged membrane [20]. Substitution of Ala (less hydrophobic and less bulky than Val) for Val on the hydrophilic face of VA1 possibly facilitated closer contact between the negatively charged S. aureus membrane and the peptide, therefore enhancing the electrostatic effect and resulting in increased peptide activity. As there was no alteration on the hydrophobic face, there was no change in activity against E. coli and eukaryotic
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cells. In VA2, the above substitution, along with one more Ala substitution for Trp on the hydrophobic face, drastically reduced the antibiotic potential of the peptide, suggesting the significance of Trp in determining the potency of the peptide. The significance of Trp residues in determining the potency of peptides has been reported in the literature [21]. Many potent naturally occurring AMPs such as indolicidin, melittin and tritrpticin are rich in Trp residues [22,23]. However, Trp residues are also responsible for the high haemolytic activity of these peptides. VA3, where one Ala was substituted for Val on the hydrophobic face along with one Ala at the hydrophilic face (as in VA1), also resulted in lower potency of the peptide. Lowered potency resulted from decreased hydrophobicity of the peptide [24]. Lowered hydrophobicity in VA2 and VA3 also resulted in lower haemolysis and cytotoxicity at high concentrations of peptides. Antibacterial activity of the peptides also correlated with the peptides’ secondary structure [25]. The current results demonstrated that the most helical peptide VA3 was less potent than the moderately helical peptide VA1, and the least helical peptide VA2 was also the least potent. Most naturally occurring peptides have been shown to have random structure in aqueous solutions; however, they acquire a helical structure in membranes. It has been shown that a certain degree of helicity with intrinsic structural flexibility of the peptide facilitates its interactions with the lipid membrane and helps the peptide to traverse the membrane [18]. Highly helical peptides that lack the intrinsic structural flexibility which helps the peptide to interact and span the membrane therefore might not be very effective in membrane interactions. The ability of microbes to evolve and generate mutant forms that escape drugs has led to loss of drug potency over time. A combination of drugs with different targets reduces the chances of drug resistance [26]. Moreover, relatively low doses of drugs are required when used in combination with other drugs [27]. We observed lower MIC values for peptides against E. coli and S. aureus when they were used in combination with sublethal concentrations of the antibiotic drugs kanamycin and rifampicin. The peptides either had additive or synergistic effects in combination with the drugs. Since these drugs target the protein synthesis machinery of bacteria, their synergy with membrane-active peptides will definitely be more intricate, hence making it difficult for bacteria to develop resistance. The most remarkable observation of the present work was the antiplasmodial activity of the peptides, alone and in combination with the well-known antimalarial drug CQ. Plasmodium falciparum, the species responsible for most deaths associated with malaria, has acquired resistance to all available antimalarial drugs with the exception of artemisinin derivatives [28]. In this case, peptide and drug combination therapy becomes one of the most significant strategies to combat increasing drug resistance. The current results showed that IC50 values of VA1, VA2 and VA3 were 10 M, >40 M and 15 M, respectively, against P. falciparum. However, in combination with 40 nM CQ, the IC50 values decreased four-fold for VA1 and VA2 and two-fold for VA3. In addition, the results also showed that haemolysis of parasitised RBCs was negligible even after 48 h of incubation with peptide in combination with CQ. Literature shows that there are a number of natural and engineered AMPs with antiplasmodial activity, such as cecropin, magainin, defensin, gomesin, lipodermaseptin derivatives and defensin derivative NK-2 [29,30]. However, most of them fall short of the requirements for an effective antiplasmodial molecule, primarily because they lose their effectiveness in the presence of serum proteins and simultaneously cause high haemolysis. They also suffer from poor bioavailability. The peptides designed for this study have a longer shelf-life since they harbour the unnatural amino acid dehydrophenylalanine, which makes the peptide relatively stable towards proteases.
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Furthermore, low IC50 values, non-toxicity to mammalian cells, and synergistic activity of the peptides with known drugs makes them more effective tools for battling increasing microbial drug resistance. Acknowledgments The authors are grateful to Dr Dinkar Sahal [International Centre for Genetic Engineering and Biotechnology (ICGEB), New Delhi, India] for providing strains of Escherichia coli and Staphylococcus aureus. They acknowledge M. Dinesh (Malaria Group, ICGEB) for providing P. falciparum culture and for help with the SYBR Green assay. Funding: The authors would like to thank the Council of Scientific and Industrial Research, India, for financial assistance. They acknowledge core funding from International Centre for Genetic Engineering and Biotechnology (ICGEB) (New Delhi, India). Competing interests: None declared. Ethical approval: Not required. References [1] Levy SB, Marshall B. Antibacterial resistance worldwide: causes, challenges and responses. Nat Med 2004;10(12 Suppl.):S122–9. [2] Rybak MJ, McGrath BJ. Combination antimicrobial therapy for bacterial infections. Guidelines for the clinician. Drugs 1996;52:390–405. [3] Hancock RE. Cationic peptides: effectors in innate immunity and novel antimicrobials. Lancet Infect Dis 2001;1:156–64. [4] Zasloff M. Antimicrobial peptides of multicellular organisms. Nature 2002;415:389–95. [5] Papo N, Shai Y. Host defense peptides as new weapons in cancer treatment. Cell Mol Life Sci 2005;62:784–90. [6] Gifford JL, Hunter HN, Vogel HJ. Lactoferricin: a lactoferrin-derived peptide with antimicrobial, antiviral, antitumor and immunological properties. Cell Mol Life Sci 2005;62:2588–98. [7] Pacor S, Giangaspero A, Bacac M, Sava G, Tossi A. Analysis of the cytotoxicity of synthetic antimicrobial peptides on mouse leucocytes: implications for systemic use. J Antimicrob Chemother 2002;50:339–48. [8] Rathinakumar R, Wimley WC. Biomolecular engineering by combinatorial design and high-throughput screening: small, soluble peptides that permeabilize membranes. J Am Chem Soc 2008;130:9849–58. [9] Tew GN, Liu D, Chen B, Doerksen RJ, Kaplan J, Carroll PJ, et al. De novo design of biomimetic antimicrobial polymers. Proc Natl Acad Sci USA 2002;99:5110–4. [10] Pathak S, Chauhan VS. Rationale-based, de novo design of dehydrophenylalanine-containing antibiotic peptides and systematic modification in sequence for enhanced potency. Antimicrob Agents Chemother 2011;55:2178–88. [11] Meyer WL, Kuyper LF, Lewis RB, Templeton GE, Woodhead SH. The amino acid sequence and configuration of tentoxin. Biochem Biophys Res Commun 1974;56:234–40.
[12] Mathur P, Ramakumar S, Chauhan VS. Peptide design using ␣,-dehydro amino acids: from -turns to helical hairpins. Biopolymers 2004;76:150–61. [13] Kaiser E, Colescott RL, Bossinger CD, Cook PI. Color test for detection of free terminal amino groups in the solid-phase synthesis of peptides. Anal Biochem 1970;34:595–8. [14] Smilkstein M, Sriwilaijaroen N, Kelly JX, Wilairat P, Riscoe M. Simple and inexpensive fluorescence-based technique for high-throughput antimalarial drug screening. Antimicrob Agents Chemother 2004;48:1803–6. [15] Mathur P, Jagannathan NR, Chauhan VS. ␣,-Dehydrophenylalanine containing cecropin–melittin hybrid peptides: conformation and activity. J Pept Sci 2007;13:253–62. [16] Rason MA, Randriantsoa T, Andrianantenaina H, Ratsimbasoa A, Menard D. Performance and reliability of the SYBR Green I based assay for the routine monitoring of susceptibility of Plasmodium falciparum clinical isolates. Trans R Soc Trop Med Hyg 2008;102:346–51. [17] Giangaspero A, Sandri L, Tossi A. Amphipathic ␣ helical antimicrobial peptides. Eur J Biochem 2001;268:5589–600. [18] Tossi A, Tarantino C, Romeo D. Design of synthetic antimicrobial peptides based on sequence analogy and amphipathicity. Eur J Biochem 1997;250:549–58. [19] Ghosh JK, Shaool D, Guillaud P, Ciceron L, Mazier D, Kustanovich I, et al. Selective cytotoxicity of dermaseptin S3 toward intraerythrocytic Plasmodium falciparum and the underlying molecular basis. J Biol Chem 1997;272:31609–16. [20] Zelezetsky I, Pacor S, Pag U, Papo N, Shai Y, Sahl HG, et al. Controlled alteration of the shape and conformational stability of ␣-helical cell-lytic peptides: effect on mode of action and cell specificity. Biochem J 2005;390:177–88. [21] Schibli DJ, Epand RF, Vogel HJ, Epand RM. Tryptophan-rich antimicrobial peptides: comparative properties and membrane interactions. Biochem Cell Biol 2002;80:667–77. [22] Selsted ME, Novotny MJ, Morris WL, Tang YQ, Smith W, Cullor JS. Indolicidin, a novel bactericidal tridecapeptide amide from neutrophils. J Biol Chem 1992;267:4292–5. [23] Blondelle SE, Simpkins LR, Pérez-Payá E, Houghten RA. Influence of tryptophan residues on melittin’s hemolytic activity. Biochim Biophys Acta 1993;1202:331–6. [24] Liu LP, Li SC, Goto NK, Deber CM. Threshold hydrophobicity dictates helical conformations of peptides in membrane environments. Biopolymers 1996;39:465–70. [25] Hwang PM, Vogel HJ. Structure–function relationships of antimicrobial peptides. Biochem Cell Biol 1998;76:235–46. [26] Cirioni O, Silvestri C, Ghiselli R, Orlando F, Riva A, Mocchegiani F, et al. Protective effects of the combination of ␣-helical antimicrobial peptides and rifampicin in three rat models of Pseudomonas aeruginosa infection. J Antimicrob Chemother 2008;62:1332–8. [27] Minahk CJ, Dupuy F, Morero RD. Enhancement of antibiotic activity by sub-lethal concentrations of enterocin CRL35. J Antimicrob Chemother 2004;53:240–6. [28] Wongsrichanalai C, Pickard AL, Wernsdorfer WH, Meshnick SR. Epidemiology of drug-resistant malaria. Lancet Infect Dis 2002;2:209–18. [29] Gwadz RW, Kaslow D, Lee JY, Maloy WL, Zasloff M, Miller LH. Effects of magainins and cecropins on the sporogonic development of malaria parasites in mosquitoes. Infect Immun 1989;57:2628–33. [30] Gelhaus C, Jacobs T, Andra J, Leippe M. The antimicrobial peptide NK-2, the core region of mammalian NK-lysin, kills intraerythrocytic Plasmodium falciparum. Antimicrob Agents Chemother 2008;52:1713–20.
Contents lists available at SciVerse ScienceDirect
International Journal of Antimicrobial Agents journal homepage: http://www.elsevier.com/locate/ijantimicag
In vitro antibacterial and antimalarial activity of dehydrophenylalanine-containing undecapeptides alone and in combination with drugs Sarika Pathak Sharma a , Jyotsna Sharma a,1 , Shamsher Singh Kanwar b , Virander Singh Chauhan a,∗ a b
International Centre for Genetic Engineering and Biotechnology, New Delhi 110067, India Department of Biotechnology, Himachal Pradesh University, Shimla 171005, India
a r t i c l e
i n f o
Article history: Received 11 August 2011 Accepted 6 October 2011 Keywords: Antimicrobial peptides De novo design Dehydrophenylalanine Antiplasmodial activity
a b s t r a c t A set of three cationic undecapeptides, analogous to the previously reported peptide VS2 (KWFWKFVKFVK), was created by alanine substitution in order to probe the effect of hydrophobicity on peptide activity. The activities of these peptides were determined against Escherichia coli, Staphylococcus aureus and the malaria parasite Plasmodium falciparum. VA1, the closest analogue of VS2, showed five-fold augmented activity [minimum inhibitory concentration (MIC) = 10 M] against the Gram-positive bacterium S. aureus. The designed analogues were non-haemolytic and non-cytotoxic at their MICs and clinically relevant concentrations. By alanine substitution, it was also possible to probe the critical role of tryptophan residues in determining peptide potency. Circular dichroism studies of the peptides in a membrane-mimetic system showed a correlation between peptide helicity and antimicrobial activity. The peptides were also tested in combination with sublethal concentrations of antibiotic drugs (rifampicin and kanamycin) and the antimalarial drug chloroquine. In combination with these drugs, the effect of the peptides was synergistic or additive. These results provide insight into basic design principles for generating new clinically relevant lead peptides. It also provides an alternative strategy where a peptide and a non-peptide drug can be used in combination to battle increasingly drug-resistant microbes. © 2011 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.
1. Introduction The widespread use of antibiotics in recent years has led to the development of microbial strains that are resistant to almost all existing antibiotics [1]. This generates an urgent need for either new antibiotics or an alternative strategy such as combination therapy, where a combination of two or more drugs are used for the treatment of infection [2]. In the development of newer antibiotics, host defence peptides or cationic antimicrobial peptides (AMPs) have emerged as potential therapeutic agents, primarily because the likelihood of development of resistance to them is very low [3]. In addition, AMPs possess a broad spectrum of antimicrobial activity against Gram-positive and Gram-negative bacteria, fungi and protozoa [4]. Some of them are also known to have antiviral and anticancer activities [5,6]. Despite being suitable candidates for tackling microbial resistance, clinical development of naturally occurring AMPs has been hampered because of their poor
∗ Corresponding author. Tel.: +91 11 2674 1358; fax: +91 11 2616 2316. E-mail address: [email protected] (V.S. Chauhan). 1 Present address: FB Biology, Philipps-Universität, 35032 Marburg, Germany.
bioavailability, large size and mammalian cell cytotoxicity [7]. Synthetic or designer AMPs have recently become a focus for drug design owing to high-throughput studies that generated multiple synthetic peptides active against a broad range of pathogens [8,9]. In our previous work, we have reported the de novo design of a series of AMPs with activity towards the Gram-negative bacterium Escherichia coli and the Gram-positive bacterium Staphylococcus aureus [10]. These peptides incorporated a non-natural amino acid, dehydrophenylalanine, which is the dehydro analogue of the amino acid phenylalanine and is found in the antibiotic peptides of fungal origin such as tentoxin [11]. The presence of these residues in peptides has been shown to impart relative protease stability to peptides [12]. In this study, the sequence of a previously designed undecapeptide (VS2) was optimised in terms of high antibacterial activity and low eukaryotic cell toxicity. Three undecapeptides analogous to VS2 were generated and their biological activities were studied. Here we describe the in vitro activities of these peptides against E. coli and S. aureus (meticillin-resistant). We also show the increased effectiveness of these peptides at low concentrations when they are used in combination with the antibiotics kanamycin and rifampicin. Since these peptides were expected to have no or negligible haemolysis, their activity against the malaria
0924-8579/$ – see front matter © 2011 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved. doi:10.1016/j.ijantimicag.2011.10.008
S.P. Sharma et al. / International Journal of Antimicrobial Agents 39 (2012) 146–152
parasite Plasmodium falciparum was also determined, both independently and in combination with a sublethal concentration of the conventional antimalarial drug chloroquine (CQ). 2. Materials and methods 2.1. Materials Bacterial strains E. coli ML35p and S. aureus ATCC 700699 were kind gifts from Dr Dinkar Sahal [International Center for Genetic Engineering and Biotechnology (ICGEB), New Delhi, India]. Amino acid derivatives and resin for peptide synthesis were obtained from Novabiochem (Merck, Darmstadt, Germany). Diisopropylcarbodiimide (DIPCDI), piperidine, dimethylformamide (DMF), dichloromethane, hydroxybenzotriazole (HOBt), isobutyl chloroformate, trifluoroacetic acid (TFA), triisopropylsilane, dl-threo--phenylserine, sodium hydroxide, citric acid, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), dimethyl sulphoxide (DMSO), bovine serum albumin (BSA) and N-methylmorpholine were from Sigma-Aldrich (St Louis, MO). RPMI 1640 and fetal bovine serum (FBS) were from Invitrogen (Carlsbad, CA). Sodium chloride, acetic anhydride and tetrahydrofuran were from Qualigens (Mumbai, India). Ethyl acetate, diethyl ether, sodium acetate and sodium sulphate were from Merck (Mumbai, India). Silica gel thin-layer chromatography plates (60F-254) were from Merck. Acetic acid was from SD Fine-Chem Ltd. (Mumbai, India). Acetonitrile was from Burdick and Jackson (Muskegon, MI). 2.2. Peptide synthesis Peptides were synthesised as C-terminal amides using standard Fmoc chemistry on rink amide MBHA (4-methylbenzhydrylamine hydrochloride salt) resin in the manual mode, with DIPCDI and HOBt as coupling agents. Peptides were acetylated at the Nterminus and amidated at the C-terminus. Both the coupling of amino acids and the Fmoc deprotection were monitored by the Kaiser test [13]. The amino acid dehydrophenylalanine (Phe) was introduced into peptides as an Fmoc-X-Phe azalactone [where X is Lys (Boc), Trp (Boc) or Ala] dipeptide block, which was allowed to couple overnight in DMF [10,12]. 2.3. Peptide purification and mass spectrometry Crude peptides were purified by reverse-phase highperformance liquid chromatography using a water–acetonitrile gradient on a C18 Phenomenex column (2 × 15 cm, 15 mm column length, flow rate 5 mL/min; Shimadzu, Kyoto, Japan); gradient of 5% to 65% acetonitrile and 0.1% TFA for 70 min, with detection at 214 nm and 280 nm. The identity of the highly purified (95%) peptides was confirmed by electrospray ionisation mass spectrometry performed at the ICGEB. Concentrations of the peptide stocks were determined by measurement of the absorbance at 280 nm [ 280, 19 000 M−1 cm−1 for ␣,-didehydrophenylalanine (Phe) and 5050 M−1 cm−1 for tryptophan]. 2.4. Antibiotic susceptibility testing Escherichia coli and S. aureus were grown overnight and were diluted in Mueller–Hinton broth (DifcoTM ; Becton Dickinson, Sparks, MD) to a cell density of 105 colony-forming units/mL. Then, 100 L of this culture was aliquoted into the wells of a 96well flat-bottomed microtitre plate (Costar; Corning, Acton, MA) and 11 L of 10X stock of each peptide concentration (in 0.2% BSA and 0.01% acetic acid) was added. Three wells were used for each concentration. The plate was incubated at 37 ◦ C in a
147
rotary shaker incubator (Kühner, Basel, Switzerland) set at 180 rpm. After 18 h of incubation, the optical density at 600 nm (OD600 ) was measured using a microtitre plate reader (VersaMaxTM tunable microplate reader; Molecular Devices, Sunnyvale, CA). The minimum inhibitory concentration (MIC) was defined as the lowest concentration of drug that inhibited measurable growth of an organism following overnight incubation. Each set of experiments was repeated at least twice. The MICs of peptides in combination with the drugs kanamycin and rifampicin (Sigma-Aldrich, St Louis, MO) at the concentration that inhibits 50% of microorganism growth (MIC50 ) were also determined by the above protocol. Peptides in combination with 2 M kanamycin (MIC50 ) were used against E. coli, whereas peptides combined with 500 nM rifampicin (MIC50 ) were used against S. aureus.
2.5. Haemolytic activity testing Human blood in 10% citrate–phosphate–dextrose was obtained from the Rotary Blood Bank (New Delhi, India). Red blood cells (RBCs) were harvested by spinning (1000 × g, 5 min, room temperature). RBCs were washed three to five times with phosphate-buffered saline (PBS). The packed cell volume obtained was used to make a 0.8% (v/v) suspension in PBS. Then, 100 L of this RBC suspension was transferred to each well of a 96-well microtitre plate and was mixed with 100 L of peptide solution at twice the desired concentration. The microtitre plate was incubated (37 ◦ C, 60 min) and centrifuged (1000 × g, 5 min, room temperature). The supernatant (100 L) was transferred to new wells and the OD414 was measured with a microtitre plate reader to monitor RBC lysis. Cells incubated with PBS alone acted as the negative control, and RBCs lysed using 0.1% Triton X-100 were used to measure 100% lysis.
2.6. Mammalian cell cytotoxicity Cytotoxicity of the antibiotic peptides was determined using the MTT assay against HeLa and fibroblast L929 cells. Briefly, cells (104 cells/well) were cultured at 37 ◦ C overnight in RPMI 1640 containing 10% FBS in 96-well microtitre plates. The next day, peptides (prepared in RPMI 1640) at concentrations of 1X and 5X MIC values were added to the cells and were incubated for 18 h at 37 ◦ C. DMSO (10%) was taken as the positive control and untreated cells served as the negative control. Then, 20 L of MTT solution (5 mg/mL) in PBS was added and the cells were incubated at 37 ◦ C for 3–4 h. Supernatant (120 L) was removed, DMSO (100 L) was added and the resulting suspension was mixed to dissolve the formazan crystals formed by MTT reduction. The ratio of OD570 for treated cells to OD570 for untreated cells was used to calculate percent viability.
2.7. Circular dichroism (CD) spectrometry CD studies were performed at 25 M peptide concentrations in 20 mM sodium dodecyl sulphate (SDS) (USB Corp., Cleveland, OH) in 10 mM phosphate buffer (pH 7.4). All spectra were recorded on a JASCO J-810 polarimeter (Jasco, Tokyo, Japan) equipped with a Peltier-type thermostat and purged continuously with dry N2 gas at 10 L/min during data acquisition. Data were collected in a quartz cuvette with path length 1 cm, between 190 nm and 340 nm at a scan speed of 50 nm/min and response time of 8 s. The average of five scans was used for analysis of the spectrum. The CD spectra of peptides were baseline-corrected by subtracting the CD spectra of SDS in buffer.
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2.8. Antiplasmodial activity of peptides 2.8.1. In vitro cultivation of Plasmodium falciparum The CQ-sensitive strain 3D7 of P. falciparum was used in in vitro blood-stage culture to test the antimalarial efficacy of the peptides VA1, VA2 and VA3 alone and in combination with 40 nM CQ. Plasmodium falciparum 3D7 cultures were maintained in fresh O-positive human erythrocytes suspended at 4% haematocrit in RPMI 1640 (Sigma) containing 0.2% sodium bicarbonate, 0.5% AlbuMAX (Sigma-Aldrich), 45 g/L hypoxanthine (Sigma-Aldrich) and 50 g/L gentamicin (Sigma-Aldrich) and incubated at 37 ◦ C under a gas mixture of 5% O2 , 5% CO2 and 90% N2 . 2.8.2. Peptide and drug dilutions Peptide and CQ stocks were prepared in Milli-Q water (Millipore, Billerica, MA) and were serially diluted with Phenol red-free RPMI 1640 to achieve the required concentrations. When used in combination, a fixed concentration (40 nM) of CQ was added to each serially diluted peptide concentration. CQ and test peptides were then placed in 96-well flat-bottom tissue culture-grade plates. 2.8.3. Assay for antiplasmodial activity and haemolysis of parasitised red blood cells A SYBR Green® -based fluorescence assay was set-up as previously described [14]. Sorbitol-synchronised parasites were incubated under normal culture conditions at 2% haematocrit and 1% parasitaemia in the absence or presence of increasing concentrations of peptide alone or in combination with CQ. CQ was also used as a positive control, whilst 0.2% Triton X-100 was used as the negative control. After 48 h of incubation, plates were spun at 2500 × g for 10 min at 25 ◦ C. Then, 100 L of the supernatant was taken and transferred to another plate for determination of haemolysis at 414 nm. In brief, 100 L of SYBR Green I solution (Invitrogen) (0.2 L of 10,000 X SYBR Green I/mL) in lysis buffer [20 mM Tris (pH 7.5), 5 mM ethylene diamine tetra-acetic acid, 0.008% (w/v) saponin and 0.08% (v/v) Triton X-100] was added to each well, mixed gently and incubated in the dark at 37 ◦ C for 1 h. Fluorescence was measured with a Victor fluorescence multiwell plate reader (Perkin-Elmer, Waltham, MA) with excitation and emission wavelength bands centred at 485 nm and 530 nm, respectively. The fluorescence counts were plotted against the drug concentration, and the 50% inhibitory concentration (IC50 ) was determined by analysis of dose–response curves. 3. Results 3.1. Peptide analogue design In this study, three new AMPs analogous to our previously reported lead broad-spectrum undecapeptide VS2 were designed and synthesised. The analogues were identical to VS2 in length and net positive charge (four lysine residues); however, they had alanine substitutions at one or two places on the hydrophobic face. Similar to VS2, these analogues also harboured three F residues on the hydrophobic face (Table 1). Fig. 1 shows a helical wheel representation of the basic peptide sequence and positions of Ala substitutions. VA1 had a single Ala substitution for Val at position 10. In VA2, two Ala were substituted, one for a Trp at position 2 and another for Val at position 10. In VA3, both the Val residues (positions 7 and 10) were replaced by Ala. 3.2. Antibacterial activity of peptides The peptide analogues VA1 and VA3 retained their antibacterial activity in the low micromolar range both against Gram-negative E. coli and Gram-positive S. aureus (Table 2). VA1 showed a MIC
Fig. 1. Basic template of the peptide analogues. (A) A 310 helical wheel representation of the peptides showing a hypothetical segregation of the hydrophobic and hydrophilic faces. White box, Ala substitution at this position was well tolerated; grey box, there was a three-fold decrease in the activity against Escherichia coli, however activity against Staphylococcus aureus was moderately improved; black box, Ala substitution at this position drastically reduced the potency of the peptide. (B) Chemical structure of the amino acid ␣,-dehydrophenylalanine (F).
of 4 M against E. coli and 10 M against S. aureus, whereas VA3 showed a MIC of 15 M for E. coli and 35 M for S. aureus. Compared with VS2, no gain in the activity of VA1 and VA3 against E. coli was observed. However, VA1 showed a five-fold lower MIC against S. aureus and VA3 also showed a reduced MIC value for S. aureus compared with VS2. VA2 was found to be only moderately active against E. coli (MIC = 75 M) and had no activity against S. aureus up to 125 M. 3.3. Antibiotic activity of peptides in combination with kanamycin and rifampicin The antibacterial activity of peptides in combination with known antibiotic drugs against S. aureus and E. coli was also tested and it was demonstrated that these peptides acted synergistically with the antibiotic drugs (Fig. 2). 3.3.1. Activity against Escherichia coli in combination with a sublethal concentration of kanamycin Since kanamycin has a MIC of ca. 4 M against E. coli ML35p, 2 M kanamycin (MIC50 ) was used in combination with different concentrations of peptides. The results show that VA1 and VA3 showed only a moderate decrease in MIC (additive effect) in combination with drug, whilst VA2 showed a very good synergy since its MIC value decreased from 75 M to 30 M. Killing curves of peptides alone and in combination with kanamycin show that the growth of the bacteria was inhibited at very early stages when the peptide was used in combination (Fig. 2A). 3.3.2. Activity against Staphylococcus aureus in combination with a sublethal concentration of rifampicin Rifampicin, a non-peptide antibiotic, has a MIC of 1 M against S. aureus [10]. An additive effect was also observed against S. aureus when VA1 and VA3 were used in combination with 500 nM rifampicin (MIC50 ). The MIC of VA1 decreased from 10 M to 7.5 M and that of VA3 decreased from 35 M to 30 M. However, VA2 again showed very good synergy with the drug. In
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149
Table 1 Amino acid sequences of peptides. Residues
Charge
F
Ala
MW (Da)
Ac-K-W-F-W-K-F-V-K-F-V-K-NH2
11
+4
3
0
1575
Ac-K-W-F-W-K-F-V-K-F-A-K-NH2 Ac-K-A-F-W-K-F-V-K-F-A-K-NH2 Ac-K-W-F-W-K-F-A-K-F-A-K-NH2
11 11 11
+4 +4 +4
3 3 3
1 2 2
1549 1433 1521
Peptide sequence VS2 Alanine analogues VA1 VA2 VA3
MW, molecular weight.
Table 2 Minimum inhibitory concentrations (MICs) of peptides against the Gram-positive bacterium Staphylococcus aureus and the Gram-negative bacterium Escherichia coli, and 50% inhibitory concentrations (IC50 ) of peptides against the malaria parasite Plasmodium falciparum alone and in combination with 40 nM chloroquine (CQ). Peptide/Drug
MIC (M)
IC50 against P. falciparum (M)
E. coli ML35p VS2 VA1 VA2 VA3 Rifampicin Kanamycin
5 4 75 15 5 4
50 10 >125 35 1 7.5
Peptide alone
In combination with CQa
N/D 10 >40 15 – –
N/D 2.5 10 7.5 – –
40 nM is the IC50 value of CQ for P. falciparum 3D7 strain.
0.8
0.8
E. coli (ML35p)
0.7 0.6 OD 600
S. aureus (ATCC 700699)
0.7 0.6
VA1
0.5 VA1+Kanamycin (2μM)
0.4 0.3
OD 600
a
S. aureus ATCC 700699
VA1
0.5
VA1+ Rifampicin (500 nM)
0.4 0.3
0.2
0.2
0.1
0.1
0
0 0
1
2
3
4
5
10
0
1
0.8
0.5
15
20
S. aureus (ATCC 700699)
0.6
VA2
VA2+Kanamycin (2μM)
0.5
VA2+Rifampicin (500nM)
0.4 0.3
0.4 0.3
0.2
0.2
0.1
0.1 0
0 0
5
10
20
30
40
0
50
5
10
Peptide conc (μM) E. coli (ML35p)
0.8
VA3
0.7
0.6
VA3+ Kanamycin (2μM)
0.6
25 50 75 Peptide conc (μM)
0.4 0.3
VA3+Rifampicin (500nM)
0.4 0.3
0.2
0.2
0.1
0.1
0
125
VA3
0.5
0.5
100
S. aureus (ATCC 600799)
0.8
0.7
OD 600
OD 600
12.5
VA2 OD 600
OD 600
0.8 0.7
0.6
10
Peptide conc (μM)
E. coli (ML35p)
0.7
7.5
5
Peptide conc (μM)
0 0
0.5
1
2.5
5
Peptide conc (μM)
(A)
10
15
0
1
5
7.5
10 12.5 15
20
25
30
35
Peptide conc (μM)
(B)
Fig. 2. Antibacterial activities of peptides alone and in combination with antibacterial drugs at a concentration that inhibits 50% of microorganism growth (MIC50 ). (A) Growth inhibition curve of Escherichia coli ML35p treated with peptide alone and in combination with 2 M kanamycin (MIC50 ). (B) Growth inhibition curve of Staphylococcus aureus ATCC 700699 treated with peptide alone and in combination of 500 nM rifampicin (MIC50 ). OD600 , optical density at 600 nm.
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S.P. Sharma et al. / International Journal of Antimicrobial Agents 39 (2012) 146–152
(A)
90
VA1
VA1 2.25
VA2
80
VA3
70
1.75
60
θx105 (deg cm2 mol-1
% Hemolysis
VA2
VA3
50 40 30 20
1.25
0.75
0.25 10 0
-0.25 1μM
5μM
10μM
25μM
50μM
100μM
190
210
230
250 270 Wavelength (nm)
290
310
330
Peptide conc -0.75
(B)
HeLa cell cytotoxicity
120
VA1 VA2
% Viability
100
Fig. 4. Circular dichroism spectra of peptides at 25 M in 20 mM sodium dodecyl sulphate in phosphate buffer (pH 7.4).
VA3
80
haemolysis, respectively, and VA3 showed 15% and 20% haemolysis. VA2 was least haemolytic at 100 M and showed only 6% haemolysis (Fig. 3A). Similarly, in the case of HeLa and fibroblast L929 cells, no significant cytotoxicity was observed for any peptide at MIC values (Fig. 3B and C). However, at 50 M concentration they were 10–20% cytotoxic and at 100 M they were 30–50% cytotoxic. Only VA1 showed 40–50% toxicity at 25 M and was toxic above that concentration.
60
40
20
0 5μM
10μM
25μM
50μM
100μM
3.5. Secondary structure of peptides
Peptide conc
(C)
Fibroblast L929 cell cytotoxicity 120
VA1 VA2 VA3
100
% Viability
80
60
40
CD studies show that the peptides acquired helical structures in a membrane-mimetic environment of 20 mM SDS (Fig. 4). All of the peptides exhibited an excitonic couplet at 267 nm (+) and 298 nm (−), which is the signature for the formation of a right-handed 310 helix [15] (Fig. 4). VA3 was found to be most helical (measured in terms of band intensity at 267 nm), followed by VA1 and VA2 (least helical). Correlation was also observed between helicity and activity of the peptides. The least helical peptide VA2 was also poor in potency. Increased helicity resulted in increased potency of the peptide in VA1. However, further increase in helicity resulted in lower potency of VA3 compared with VA1.
20
0 5μM
10μM
25μM
50μM
100μM
Peptide conc Fig. 3. (A) Percent haemolysis of human red blood cells after 1 h of incubation with peptides. Haemolysis by 0.2% Triton X-100 was taken as 100%. (B and C) Percent viability of HeLa cells (B) and fibroblast L929 cells (C) after 24 h of incubation with peptides in an MTT-based cell viability assay. Dimethyl sulphoxide (DMSO) (10%) was taken as control for 100% cytotoxicity.
combination, its MIC was reduced to 75 M against S. aureus whereas alone it showed no activity up to 125 M (Fig. 2B). 3.4. Cell selectivity: haemolysis and cytotoxicity to HeLa and L929 cells None of the peptides showed noteworthy haemolysis or cytotoxicity to HeLa and L929 fibroblast cells at their respective MICs (Fig. 3). However, at higher concentrations (≥50 M), VA1 and VA3 showed some degree of haemolysis and cytotoxicity. At 50 M and 100 M concentrations, VA1 showed 30% and 75%
3.6. Antiplasmodial activity of peptides alone and in combination with chloroquine Since many natural and synthetic AMPs have been shown to have antimalarial activity, the antiplasmodial activity of the designed peptides was also tested, independently and in combination with the antimalarial drug CQ using a standard SYBR Green assay (Table 2). SYBR Green is a DNA-binding cell-permeant fluorescent dye with excitation/emission at 485/535 nm [16]. The results obtained with this assay showed IC50 values of 10 M, >40 M and 15 M for VA1, VA2 and VA3, respectively. It was also shown that in combination with 40 nM CQ the IC50 values of the peptides decreased to 2.5 M, 10 M and 7.5 M for VA1, VA2 and VA3, respectively. Furthermore, haemolysis of parasitised RBCs after 48 h of incubation with peptide in combination with CQ was determined in the same experimental assay. As shown in Fig. 5, no significant haemolysis was observed even after 48 h. These results suggest that the peptides showed synergy with CQ, and such combinations can be extremely useful against the development of alarmingly increasing CQ-resistant strains of P. falciparum.
% Haemolysis of parasitised RBCs
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VA1 VA2 VA3
15
10
5
0
Peptide conc+Cq conc Fig. 5. Percent haemolysis of parasitised red blood cells incubated with peptides and 40 nM chloroquine for 48 h. Haemolysis by 0.2% Triton X-100 was taken as 100%.
4. Discussion We have previously reported rational-based design of short AMPs that act by increasing bacterial membrane permeability [10]. However, despite being potent against bacteria, some of them were non-specific in their activity and also showed RBC haemolysis and mammalian cell cytotoxicity. In that work, we had optimised the sequence of peptide VS2 in terms of potency, non-cytotoxicity and faster killing kinetics. However, we also reported that at high concentrations (well above the MIC for E. coli) the peptide caused haemolysis and cytotoxicity and required further improvement in the design in order to be a suitable drug candidate. Literature shows that the lack of cell selectivity is related to high hydrophobicity and high amphipathicity of the peptides [17]. Nevertheless, a certain degree of amphipathicity and hydrophobicity is crucial for interaction with negatively charged microbial membranes [18]. In an effort to improve peptide specificity, we tried to alter the peptide hydrophobicity and amphipathicity by replacing more hydrophobic and bulky residues (Val and Trp, respectively) present on the hydrophobic face of the peptides with a moderately hydrophobic residue Ala. We tried to reduce the overall hydrophobicity of the peptide using the following designs: replacement of single Val with Ala (VA1); replacement of one Trp and one Val each with Ala (VA2); and replacement of two Val with two Ala (VA3). This study on Ala analogues showed that reduced hydrophobicity indeed resulted in altered cell selectivity of peptides. In VA1, replacement of a single Val by Ala at the hydrophilic face only slightly lowered the hydrophobicity of the peptide. This substitution did not alter the activity of the peptide against E. coli and eukaryotic cells. However, it enhanced its potency five-fold against Gram-positive S. aureus. The augmented activity against S. aureus can be explained by the difference in the membrane compositions of Gram-negative and Gram-positive bacteria and the eukaryotic membrane. The eukaryotic membrane is zwitterionic, whereas the membrane of Gram-negative bacteria is negatively charged and partially zwitterionic and that of Gram-positive bacteria is completely negatively charged [19]. Literature shows that increased hydrophobicity of peptides increases their interaction with zwitterionic membranes (eukaryotic and Gram-negative membranes) but not with the negatively charged membrane [20]. Substitution of Ala (less hydrophobic and less bulky than Val) for Val on the hydrophilic face of VA1 possibly facilitated closer contact between the negatively charged S. aureus membrane and the peptide, therefore enhancing the electrostatic effect and resulting in increased peptide activity. As there was no alteration on the hydrophobic face, there was no change in activity against E. coli and eukaryotic
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cells. In VA2, the above substitution, along with one more Ala substitution for Trp on the hydrophobic face, drastically reduced the antibiotic potential of the peptide, suggesting the significance of Trp in determining the potency of the peptide. The significance of Trp residues in determining the potency of peptides has been reported in the literature [21]. Many potent naturally occurring AMPs such as indolicidin, melittin and tritrpticin are rich in Trp residues [22,23]. However, Trp residues are also responsible for the high haemolytic activity of these peptides. VA3, where one Ala was substituted for Val on the hydrophobic face along with one Ala at the hydrophilic face (as in VA1), also resulted in lower potency of the peptide. Lowered potency resulted from decreased hydrophobicity of the peptide [24]. Lowered hydrophobicity in VA2 and VA3 also resulted in lower haemolysis and cytotoxicity at high concentrations of peptides. Antibacterial activity of the peptides also correlated with the peptides’ secondary structure [25]. The current results demonstrated that the most helical peptide VA3 was less potent than the moderately helical peptide VA1, and the least helical peptide VA2 was also the least potent. Most naturally occurring peptides have been shown to have random structure in aqueous solutions; however, they acquire a helical structure in membranes. It has been shown that a certain degree of helicity with intrinsic structural flexibility of the peptide facilitates its interactions with the lipid membrane and helps the peptide to traverse the membrane [18]. Highly helical peptides that lack the intrinsic structural flexibility which helps the peptide to interact and span the membrane therefore might not be very effective in membrane interactions. The ability of microbes to evolve and generate mutant forms that escape drugs has led to loss of drug potency over time. A combination of drugs with different targets reduces the chances of drug resistance [26]. Moreover, relatively low doses of drugs are required when used in combination with other drugs [27]. We observed lower MIC values for peptides against E. coli and S. aureus when they were used in combination with sublethal concentrations of the antibiotic drugs kanamycin and rifampicin. The peptides either had additive or synergistic effects in combination with the drugs. Since these drugs target the protein synthesis machinery of bacteria, their synergy with membrane-active peptides will definitely be more intricate, hence making it difficult for bacteria to develop resistance. The most remarkable observation of the present work was the antiplasmodial activity of the peptides, alone and in combination with the well-known antimalarial drug CQ. Plasmodium falciparum, the species responsible for most deaths associated with malaria, has acquired resistance to all available antimalarial drugs with the exception of artemisinin derivatives [28]. In this case, peptide and drug combination therapy becomes one of the most significant strategies to combat increasing drug resistance. The current results showed that IC50 values of VA1, VA2 and VA3 were 10 M, >40 M and 15 M, respectively, against P. falciparum. However, in combination with 40 nM CQ, the IC50 values decreased four-fold for VA1 and VA2 and two-fold for VA3. In addition, the results also showed that haemolysis of parasitised RBCs was negligible even after 48 h of incubation with peptide in combination with CQ. Literature shows that there are a number of natural and engineered AMPs with antiplasmodial activity, such as cecropin, magainin, defensin, gomesin, lipodermaseptin derivatives and defensin derivative NK-2 [29,30]. However, most of them fall short of the requirements for an effective antiplasmodial molecule, primarily because they lose their effectiveness in the presence of serum proteins and simultaneously cause high haemolysis. They also suffer from poor bioavailability. The peptides designed for this study have a longer shelf-life since they harbour the unnatural amino acid dehydrophenylalanine, which makes the peptide relatively stable towards proteases.
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Furthermore, low IC50 values, non-toxicity to mammalian cells, and synergistic activity of the peptides with known drugs makes them more effective tools for battling increasing microbial drug resistance. Acknowledgments The authors are grateful to Dr Dinkar Sahal [International Centre for Genetic Engineering and Biotechnology (ICGEB), New Delhi, India] for providing strains of Escherichia coli and Staphylococcus aureus. They acknowledge M. Dinesh (Malaria Group, ICGEB) for providing P. falciparum culture and for help with the SYBR Green assay. Funding: The authors would like to thank the Council of Scientific and Industrial Research, India, for financial assistance. They acknowledge core funding from International Centre for Genetic Engineering and Biotechnology (ICGEB) (New Delhi, India). Competing interests: None declared. Ethical approval: Not required. References [1] Levy SB, Marshall B. Antibacterial resistance worldwide: causes, challenges and responses. Nat Med 2004;10(12 Suppl.):S122–9. [2] Rybak MJ, McGrath BJ. Combination antimicrobial therapy for bacterial infections. Guidelines for the clinician. Drugs 1996;52:390–405. [3] Hancock RE. Cationic peptides: effectors in innate immunity and novel antimicrobials. Lancet Infect Dis 2001;1:156–64. [4] Zasloff M. Antimicrobial peptides of multicellular organisms. Nature 2002;415:389–95. [5] Papo N, Shai Y. Host defense peptides as new weapons in cancer treatment. Cell Mol Life Sci 2005;62:784–90. [6] Gifford JL, Hunter HN, Vogel HJ. Lactoferricin: a lactoferrin-derived peptide with antimicrobial, antiviral, antitumor and immunological properties. Cell Mol Life Sci 2005;62:2588–98. [7] Pacor S, Giangaspero A, Bacac M, Sava G, Tossi A. Analysis of the cytotoxicity of synthetic antimicrobial peptides on mouse leucocytes: implications for systemic use. J Antimicrob Chemother 2002;50:339–48. [8] Rathinakumar R, Wimley WC. Biomolecular engineering by combinatorial design and high-throughput screening: small, soluble peptides that permeabilize membranes. J Am Chem Soc 2008;130:9849–58. [9] Tew GN, Liu D, Chen B, Doerksen RJ, Kaplan J, Carroll PJ, et al. De novo design of biomimetic antimicrobial polymers. Proc Natl Acad Sci USA 2002;99:5110–4. [10] Pathak S, Chauhan VS. Rationale-based, de novo design of dehydrophenylalanine-containing antibiotic peptides and systematic modification in sequence for enhanced potency. Antimicrob Agents Chemother 2011;55:2178–88. [11] Meyer WL, Kuyper LF, Lewis RB, Templeton GE, Woodhead SH. The amino acid sequence and configuration of tentoxin. Biochem Biophys Res Commun 1974;56:234–40.
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