A 17-mer Membrane-Active MSI-78 Derivative with Improved

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A 17-mer membrane-active MSI-78 derivative with improved selectivity towards bacterial cells Claudia Monteiro, Marina Pinheiro, Mariana Fernandes, Sílvia Maia, Catarina L. Seabra, Frederico Ferreira-da-Silva, Salette Reis, Paula Gomes, and M. Cristina L. Martins Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.5b00113 • Publication Date (Web): 11 Jun 2015 Downloaded from http://pubs.acs.org on June 15, 2015

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Molecular Pharmaceutics

1

A 17-mer membrane-active MSI-78 derivative with

2

improved selectivity towards bacterial cells

3

Claudia Monteiro1,2‡, Marina Pinheiro3‡, Mariana Fernandes1,2, Sílvia Maia4, Catarina L.

4

Seabra1,2,5,6, Frederico Ferreira-da-Silva1,7, Salette Reis3, Paula Gomes4, M. Cristina L.

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Martins1,2,5

6 7

No benefit of any kind will be received either directly or indirectly by the authors

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1 - I3S, Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Portugal

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2 - INEB, Instituto de Engenharia Biomédica, Universidade do Porto, Rua do Campo Alegre,

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823, 4150-180 Porto, Portugal

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3 - UCIBIO, Requimte, Faculdade de Farmácia, Universidade do Porto, Rua de Jorge Viterbo

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Ferreira 228, 4050-313 Porto, Portugal

14

4 - UCIBIO, Requimte, Departamento de Química e Bioquímica, Faculdade de Ciências,

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Universidade do Porto, Rua do Campo Alegre 687, 4169-007 Porto, Portugal

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5 - ICBAS, Instituto de Ciências Biomédicas Abel Salazar, Universidade do Porto, Rua de Jorge

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Viterbo Ferreira 228, 4050-313 Porto, Portugal

ACS Paragon Plus Environment

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6 - IPATIMUP - Institute of Molecular Pathology and Immunology of the University of Porto,

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Rua Dr. Roberto Frias, 4200-465 Porto, Portugal

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7 - IBMC-Instituto de Biologia Celular e Molecular, Unidade de Produção e Purificação de

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Proteínas, Universidade do Porto, Rua do Campo Alegre, 823, 4150-180 Porto, Portugal

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KEYWORDS

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Antimicrobial peptides, Pexiganan, MSI-78, antibiotic resistance, cytotoxicity and membrane

25

models

26

27

ABSTRACT

28

Antimicrobial peptides are widely recognized as an excellent alternative to conventional

29

antibiotics. MSI-78, a highly effective and broad spectrum AMP is one of the most promising

30

AMP for clinical application. In this study, we have designed shorter derivatives of MSI-78 with

31

the aim of improving selectivity while maintaining antimicrobial activity. Shorter 17-mer

32

derivatives were created by truncating MSI-78 at the N-and/or C-termini, while spanning MSI-

33

78 sequence. Despite the truncations made, we found a 17-mer peptide, MSI-78(4-20)

34

(KFLKKAKKFGKAFVKIL), which demonstrated to be as effective as MSI-78 against the

35

Gram-positive Staphylococcus strains tested and the Gram-negative Pseudomonas aeruginosa.

36

This shorter derivative is more selective towards bacterial cells as it was less toxic to

37

erythrocytes than MSI-78, representing an improved version of the lead peptide. Biophysical

38

studies support a mechanism of action for MSI-78(4-20) based on the disruption of the bacterial


2

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membrane permeability barrier, which in turn leads to loss of membrane integrity and ultimately

40

to cell death. These features point to a mechanism of action similar to the one described for the

41

lead peptide MSI-78.

42 43

INTRODUCTION

44

Antibiotic resistance is becoming a huge concern, with recent reports from the World Health

45

Organization (WHO) showing worrying situations worldwide 1. Methicillin resistant S. aureus

46

(MRSA) is one of the bacterial strains that causes most alarm, being estimated that 25% of S.

47

aureus isolates in the USA are resistant while this percentage can be even higher in other

48

countries 2. Resistance is also a problem in Gram-negative bacteria, such as in the case of multi-

49

resistant Pseudomonas aeruginosa, with statistics revealing that in some countries multi-resistant

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Pseudomonas represent up to 45% of the isolates 3.

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The increasing emergency to combat resistant bacterial strains has prompted the development of

52

new generations of antimicrobial agents, with antimicrobial peptides (AMP) being one of the

53

most promising alternatives 4-7.

54

AMP are ubiquitous in nature, usually 10-50 residues in length, and have the ability to interact

55

with bacterial cell membranes most often leading to membrane disruption 8-10. As their mode of

56

action does not involve specific interactions, bacteria are less prone to acquire resistance to AMP

57

11

58

identified 11, 12.

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Although AMP are promising antibiotic alternatives, their low stability and high toxicity prevent

60

efficient therapeutic application

. However, some resistance mechanisms that involve alterations of the cell wall have been

13

. Therefore, improving AMP potency and selectivity towards


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bacterial cells is needed. AMP selectivity is mainly determined by charge and hydrophobicity.

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While net charge is crucial in the establishment of electrostatic interactions between the

63

positively charged peptide and the negatively charged lipid head group of the bacterial lipid

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bilayer, hydrophobicity is determinant in the insertion of the AMP into the lipid bilayer 14.

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Another concern for bringing AMP into therapeutic applications is the high cost of production

66

associated with peptide synthesis. Therefore, developing short AMP with higher bacterial cell

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selectivity is the purpose of this study

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cytotoxicity, for example a 15 residue shortened melitin and a shorter derivative of HP(2-20)

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were 300 times less toxic to erythrocytes than their parent peptides 15-17.

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MSI-78 is a well-studied AMP, commercially known as Pexiganan, developed for the treatment

71

of infected diabetic foot ulcers

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effective against Gram-positive and Gram-negative bacteria 18, 20. MSI-78 belongs to the class of

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α-helical AMPs and it has been described to form dimers stabilized by hydrophobic interactions

74

21

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membranes via toroidal-type pore formation 20. Being one of the best-studied and efficient AMP,

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MSI-78 is an excellent lead peptide to create shorter derivatives.

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In this work, we aimed at creating a shorter MSI-78 derivative while maintaining antimicrobial

78

activity. We identified a 17-mer peptide, MSI-78(4-20), which demonstrated to be as effective as

79

MSI-78 against all strains tested, despite the truncations made.

80

With the help of model biomembrane systems we unraveled the mechanism of action of MSI-

81

78(4-20). Model membranes composed of different phospholipids that mimic bacterial and

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mammalian cell membrane were chosen as they are excellent models to characterize the

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interactions between membrane active compounds and bacterial or mammalian membranes. To

18, 19

13

. In fact, it has been reported that AMP length affects

. This peptide has a broad spectrum of activity, being

. Moreover, it is known that the MSI-78 mechanism of action involves disruption of bacterial


4

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mimic the mammalian membrane 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC) was

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chosen as it is widely used

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oleoylphosphatidilcholine

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(POPG) at 1:1 molar ratio, was chosen as it has been described as one of the most biologically

88

relevant model membrane for studying the S. aureus strain 24.

22, 23

. To mimic the bacterial membrane, a mixture of 1-palmitoyl-2-

(POPC)

and

1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine

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EXPERIMENTAL SECTION

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Reagents

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Nα-Fmoc-protected amino acids, Rink amide AM resin, and 2-(1H-Benzotriazole-1-yl)-1,1,3,3-

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tetramethyluronium hexafluorophosphate (HBTU) for solid phase peptide synthesis (SPPS) were

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from NovaBiochem-EMD4Biosciences (Darmstadt, Germany). N-ethyl-N,N-diisopropylamine

95

(DIEA), 1-hydroxybenzotriazole (HOBt), trifluoroacetic acid (TFA), triisopropylsilane (TIS),

96

piperidine and all solvents for SPPS were from Sigma-Aldrich (St. Louis, MO, USA).

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Octadecylsilane stationary phase 238TPB1520 for peptide purification by medium-pressure

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reverse-phase liquid chromatography (MP-RPLC) was from Vydac (Hesperia, CA, USA). The

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lipids, 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 2-oleoyl-1-palmitoyl-sn-glycero-

100

3-phosphocholine

(POPC)

and

2-oleoyl-1-palmitoyl-sn-glycero-3-phospho-rac-(1-glycerol)

101

sodium salt (POPG) with a purity grade > 98% were also purchased from Sigma-Aldrich Co.

102

Hepes buffer used in the biophysical experiments with LUVs consisted on 10 mmol L−1 N-(2-

103

hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid), the ionic strength was adjusted to 0.1 M

104

with sodium chloride and the pH to 7.4. In the circular dichroism (CD) experiments, the buffer

105

consisted of 10 mmol L−1 of sodium phosphate buffer and the ionic strength was adjusted to 0.1


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M with sodium fluoride and the pH to 7.4. Hepes and sodium phosphate buffer were supplied by

107

Sigma-Aldrich Co. (St. Louis, MO, USA). All the other chemicals were purchased from Merck.

108 109

Peptides design and synthesis

110

Peptide sequences were designed in order to obtain 17-mer peptides based on MSI-78 sequence,

111

while spanning the entire sequence with one 1 AA shifts with the aim of identifying shorter but

112

still active fragments of MSI-78 (Table 1). The 17-mer length was chosen as it is an intermediate

113

size between the 22-mer MSI-78 and the 12-mer short derivatives of MSI-78, that considerably

114

lost its spectrum of activity as previously described by us

115

truncating peptides while maintaining a length above 15 AA may preserve antimicrobial activity

116

26

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All peptides (Table 1) were prepared as C-terminal amides by standard Fmoc/tBu SPPS on a

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Liberty1 Microwave (MW) Peptide Synthesizer (CEM Corporation, Mathews, NC, USA)

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Briefly, Rink amide AM resin was pre-swelled for 15 minutes in N,N-dimethylformamide

120

(DMF) and then transferred into the MW-reaction vessel. The initial Fmoc deprotection step was

121

carried out using 20% piperidine in DMF containing 0.1 M HOBt in two MW irradiation pulses:

122

30 seconds at 24 W plus 3 minutes at 28 W, in both cases temperature being kept under 75 ºC.

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The Fmoc-protected C-terminal amino acid (Bachem, Switzerland) was then coupled to the resin,

124

using 5 molar equivalents (eq) of the Fmoc-protected amino acid in DMF (0.2 M), 5 eq of 0.5 M

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HBTU/HOBt in DMF and 10 eq of 2 M DIEA in N-methylpyrrolidone (NMP); the coupling step

126

was carried out for 5 minutes at 35 W MW irradiation, with maximum temperature kept below

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75 ºC. The remaining amino acids were sequentially coupled in the C→N direction by means of

25

. Moreover, it has been shown that

.

27

.


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similar deprotection and coupling cycles. Following completion of sequence assembly, the

129

peptides were released from the resin with concomitant removal of side-chain protecting groups,

130

by a 3 h-acidolysis at room temperature using a TFA-based cocktail containing TIS and water as

131

scavengers (TFA/TIS/H2O 95:2.5:2.5 v/v/v). Crude products were purified by MP-RPLC to a

132

purity of at least 95%, as confirmed by high-performance liquid chromatography (HPLC)

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analysis on a Hitachi-Merck LaChrom Elite system equipped with a quaternary pump, a

134

thermostated (Peltier effect) automated sampler and a diode-array detector (DAD). Pure peptides

135

were quantified by UV-absorption spectroscopy (Helios Gama, Spectronic Unicam) and their

136

molecular weights confirmed to be as expected by electrospray ionization/ion trap mass

137

spectroscopy (ESI/IT MS; LCQ-DecaXP LC-MS system, ThermoFinnigan).

138 139

Microorganisms and growth conditions

140

Microorganisms tested in this study were the following: Staphylococcus aureus (S. aureus)

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ATCC 33591 (methicillin resistant), S. aureus ATCC 25932, S. epidermidis ATCC 35984 and P.

142

aeruginosa ATCC 27853. Bacteria were grown on Trypticase Soy Agar (TSA) plates and

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Mueller-Hinton broth (MHB).

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Antimicrobial testing

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Antimicrobial activity was assessed by Minimal Inhibitory Concentration (MIC) and Minimal

147

Bactericidal Concentration (MBC). The method used to determine the MIC was the broth

148

microdilution assay in microtiter plates, described by Wiegand and co-workers

149

follows the guidelines of the two recognized organizations, CLSI and EUCAST. Bacteria were

28

. This method


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pre-cultured on TSB overnight at 37°C and 150 rpm. After washing with phosphate buffered

151

saline (PBS) bacteria were adjusted to approximately 2×105 CFU/mL in MHB and 99 µL were

152

transferred to a 96 well plate. Polypropylene microtiter plates were used to prevent binding of the

153

peptides to the walls of the wells. Peptide dilutions were prepared in acetic acid/bovine serum

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albumin (BSA) solution and peptide concentrations tested ranged from 512 µg/mL to 0.015

155

µg/mL, adjusted according to results obtained in preliminary experiments. The peptides were

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tested in a final volume of 110 µL.

157

The minimum bactericidal concentration (MBC) was determined by plating the content of the

158

first three wells where visible growth was not observed.

159 160

Hemolysis assay on human red blood cells (RBC)

161

Human blood buffy coats were obtained from healthy volunteers (Centro Hospitalar de São João,

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EPE, Porto, Portugal) and processed to obtain RBC by centrifugation over density gradient with

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Histopaque-1077 Sigma-Aldrich Co. (St. Louis, MO, USA) according to the manufacturer’s

164

instructions. After removal of the plasma upper layer, the lower layer containing RBC was

165

washed three times in PBS. The purified RBC were diluted to 6-7 x 108 cells/mL in PBS and 99

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µL were distributed in a 96 well polypropylene microtiter plate.

167

Peptide dilutions were prepared in acetic acid/BSA solution and the range of peptide

168

concentrations tested went from 512 µg/mL to 1 µg/mL. After 1 h of incubation at 37°C under

169

5% CO2, cells were centrifuged at 900 g for 10 min and the supernatant was transferred to a 96

170

well plate. The absorbance values of the released hemoglobin were determined at 450 nm using a

171

microplate reader (SynersyMx, Biotek). Untreated cells were used as negative control and cells


8

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172

treated with 0.2% Triton X-100 as positive controls. The percentage hemolysis was calculated as

173

[(sample absorbance - negative control absorbance)/(positive control absorbance - negative

174

control absorbance)] × 100.

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Preparation of cell membrane models - large unilamellar vesicles (LUVs)

177

Lipid films were formed from chloroform solution of DMPC and POPC:POPG 1:1 molar ratio

178

lipids, dried under a stream of nitrogen and left under reduced pressure for a minimum of 45

179

min, to remove all traces of the organic solvent. LUVs were prepared by the addition of the

180

buffer, followed by vortex, yielding multilamellar vesicles (MLVs). Lipid suspensions were

181

equilibrated at 37.0 ± 0.1°C for 30 min and extruded 10 times through polycarbonate filters with

182

a diameter pore of 100 nm to form LUVs, as previously described 23.

183 184

ζ-potential of LUVs

185

The ζ-potential of LUVs in the absence and in the presence of AMP was assessed through the

186

determination of the electrophoretic mobility using a ZetaPALS (Brookhaven Instruments

187

Corporation). Typically 10 runs were performed and the temperature was maintained at 37.0 ±

188

0.1°C, being the lipid concentration kept constant at 200 µM, and AMP concentration ranged

189

from 0−40 µM.

190 191

Hydrodynamic diameter

192

The hydrodynamic diameters of LUVs in the absence and in the presence of AMP were

193

determined in BI-MAS dynamic light scattering (DLS) instrument (Brookhaven Instruments,


9


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USA). Typically 6 runs (2 min each) were performed at the temperature of 37.0 ± 0.1°C, being

195

the cumulate analysis applied to scattering data to give effective diameters and polydispersity.

196 197

Circular dichroism (CD) analysis

198

The secondary structure of the peptides MSI-78(4-20) and MSI-78, was investigated using CD

199

on a JASCO J-815 spectropolarimeter, equipped with a temperature-controlled cuvette and

200

controlled by the Spectra manager software.

201

Peptide solutions were diluted in 10 mM sodium phosphate, pH 7.4, 100 mM sodium fluoride to

202

a concentration of 40 µM with and without 5 mM DMPC or POPC:POPG 1:1 molar ratio LUVs,

203

corresponding to a peptide:lipid molar ratio of 1:125.

204

Before the measurement all peptide solutions were incubated at 37°C for 30 min. Far-UV CD

205

spectra were recorded between 195 and 260 nm using a 1 mm path length cuvette. CD spectra

206

were acquired with a scanning speed of 100 nm/min, an integration time of 1 s, and using a

207

bandwidth of 1 nm. The spectra were averaged over sixteen scans and corrected by subtraction of

208

the buffer or buffer plus LUVs signal.

209

The results are expressed as the mean residue ellipticity ϴMRW, defined as ϴMRW = ϴobs

210

(0.1MRW)/(lc), where ϴobs is the observed ellipticity in millidegrees, MRW is the mean residue

211

weight, c is the concentration in mg/ml and l is the light path length in cm.

212

The mean helix content (fH) was calculated according to Luo and Baldwin, 1997 29. Briefly, fH is

213

obtained from the ϴMRW at 222 nm of each peptide (ϴ222) according to the equation: fH = (ϴ222 -

214

ϴC)/( ϴH- ϴC), where the baseline ellipticities for random coil (ϴC) and complete helix (ϴH) are

215

given by: ϴC =2220-53T and ϴH =(-44000 +250T)(1-3/N), where T is the temperature in ºC and

216

N is the chain length in number of residues.


10

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217 218

RESULTS

219

Antimicrobial activity

220

MIC of MSI-78 and its 17-mer derivatives against the Gram-positive S. aureus and S.

221

epidermidis and the Gram-negative P. aeruginosa are described in Table 2.

222

Regarding Gram-positive S. aureus strains, the 17-mer peptide MSI-78(4-20) was the most

223

effective with MIC values of 32 µg/ml for the MRSA strain and 8-16 µg/ml for the non-resistant

224

S. aureus strain. Similar MIC values were obtained with the lead peptide (MSI-78). MSI-78(1-

225

17) maintained also a high antimicrobial activity with MIC values of 64-128 µg/ml for the

226

MRSA strain and 32 µg/ml for the non-resistant S. aureus strain. The MRSA strain is slightly

227

less susceptible to the peptides than the non-resistant strain. S. epidermidis ATCC 35984 was

228

more susceptible to the MSI-78 derivatives than S. aureus with all the 17-mer peptides being

229

effective with MIC values ranging from 2-32 µg/ml. Still, MSI-78(4-20) also proved to be the

230

most effective 17-mer derivative against this strain. Results for the Gram-negative P. aeruginosa

231

demonstrate that this strain is much more susceptible to the 17-mer peptides and to MSI-78 than

232

Gram-positive Staphylococcus strains. All 17-mer peptides maintained the antibacterial effect

233

with MIC values below 8 µg/ml, being the MIC for MSI-78 exceptionally low, 0.5-1 µg/ml.

234

MSI-78(4-20) was the most effective of all 17-mer derivatives also against P. aeruginosa.

235

Overall, we found two 17-mer peptides MSI-78(4-20) and MSI-78(1-17) that maintained high

236

spectrum of activity, being effective against all strains tested, with MSI-78(4-20) standing out,

237

has it demonstrated to be equipotent to MSI-78.


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MBC results were in general within the same range or two fold higher than MIC, indicating

239

bactericidal effect of the peptides for the concentrations tested.

240

MSI-78 antimicrobial activity was in general found to be in line with previously published

241

studies. Although MSI-78 MIC values obtained for S. epidermidis and P. aeruginosa are slightly

242

lower than published data, some variation is reported in the literature for these strains 18, 20, 30.

243 244

Hemolytic activity

245

Cytotoxicity studies were performed with MSI-78 and MSI-78(4-20) as it demonstrated to be as

246

effective as the lead peptide.

247

MSI-78(4-20) was less cytotoxic to RBC than the lead AMP, MSI-78 (Fig. 1). MSI-78 showed

248

some toxicity at high concentrations (53% hemolysis at 512 µg/mL, 25% hemolysis at 256

249

µg/mL and 13% hemolysis at 128 µg/mL), which is in agreement with previously published data

250

31

251

concentrations tested (4% hemolysis at 512 µg/mL, 2% hemolysis at 256 µg/mL and 1%

252

hemolysis at 128 µg/mL).

. The 17-mer peptide, MSI-78(4-20) showed very low toxicity to RBC even at the higher

253 254

AMP interaction with membrane mimetic systems

255

ζ-potential

256

The ζ-potential of DMPC and POPC:POPG LUVs in the absence and in presence of the AMPs

257

MSI-78(4-20) and MSI-78 is represented in Fig. 2.

258

zwitterionic DMPC LUVs was as expected close to zero 32. In presence of MSI-78(4-20) or MSI-

259

78, the ζ-potential remains nearly unchanged (Fig. 2A). The addition of increased concentrations

The determined ζ-potential of the


12

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260

of either peptide to DMPC LUVs caused almost no alteration on the zeta potential values,

261

suggesting a weak or non-interaction with the mammalian membrane model. The ζ-potential of

262

POPC:POPG LUVs was as expected negative, being significantly altered by addition of either

263

AMP and becoming less negative as the concentration of MSI-78(4-20) and MSI-78 increased

264

(Fig. 2B)

265

was achieved after the addition of both peptides (Fig. 2B). However, the variation of the ζ-

266

potential values was even more evident in the case of MSI-78 for lower concentrations (below 20

267

µM) of peptide (Fig. 2B). These results suggest that the positively charged peptides interact with

268

the negatively charged POPC:POPG LUVs, inducing changes in the membrane surface charge

269

by direct peptide binding and/or insertion in the bacterial membrane model. The MSI-78 more

270

pronounced effect is probably due to the high positive charge displayed (+9) in comparison to

271

MSI-78(4-20) (+7), being consequently more prone to establish electrostatic interactions with the

272

negatively charged bacterial membrane.

33

. An overall change from approximately -28 mV to nearly neutral potential values

273 274

Hydrodynamic diameter

275

The size of DMPC and POPC:POPG LUVs in absence and presence of different concentrations

276

of the AMPs MSI-78(4-20) and MSI-78 is represented in Fig. 3. In the absence of peptides,

277

DMPC and POPC:POPG LUVs presented a size of approximately 100 nm (Fig. 3A), as expected

278

regarding their preparation. In presence of the peptides, DMPC LUVs exhibited a narrow size

279

distribution with a mean diameter of approximately 103-118 nm (Fig. 3A), pointing to a weak or

280

non-influence of both peptides in the mammalian cell membrane model even at high peptide

281

concentrations. On the other hand, peptide interactions with POPC:POPG LUVs induced a

282

pronounced increase of the LUVs size, at peptide concentrations higher than 10 µM (Fig. 3B).


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283

Moreover, this result is emphasized by the high polydispersity values obtained, indicating the

284

presence of a heterogeneous population (polydispersity > 0.1) (Fig. 3B), and pointing to the

285

existence of aggregated structures. Therefore, bacterial membrane model binding facilitates

286

nucleation-dependent AMP

287

hydrodynamic diameter and polydispersity obtained in the case of POPC:POPG LUVs for higher

288

AMP concentrations.

aggregation.

The

aggregation

explains

the

larger

mean

289 290

AMP structural analysis (Secondary Structure)

291

CD spectra of MSI-78 and MSI-78(4-20) in aqueous buffer exhibited a negative minimum at ≈

292

198 nm suggesting random coil conformations (Fig. 4A). In the presence of DMPC LUVs,

293

spectra also exhibit characteristic profiles of unordered peptides, pointing out to no toxicity

294

towards mammalian cells (Fig. 4B). In the presence of POPC:POPG (1:1 molar ratio) LUVs, two

295

negative minima at ≈ 208 nm and ≈ 222 suggest the presence of α-helical conformations (Fig.

296

4C). For all peptides, results indicate a transition from a random coil to an ordered secondary

297

structure upon interaction with the bacterial membrane mimetic system POPC:POPG LUVs. In

298

the presence of this system, calculated mean helix contents show slight differences between the

299

peptides. The lead 22-mer peptide MSI-78 has 62% helicity, whereas helicity values around 40-

300

45% have been reported by Mercke et al. for this peptide when bound to POPC small unilamellar

301

vesicles (SUV)

302

shows a difference of approximately 30% in the helix content between the two peptides.

303

Fig. 4D represents the helical wheel projections of the peptides where the relative position of the

304

amino acids and its polar/non-polar character is displayed. Table 3 summarizes the

305

physicochemical properties and structural analysis of the peptides. MSI-78(4-20) displays higher

34, 35

. For MSI-78(4-20) the determined mean helix content was 33%, which


14

Page 15 of 27

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306

hydrophobicity and higher hydrophobic moment than MSI-78. However, no hydrophobic face is

307

predicted for MSI-78(4-20) by Heliquest, as it is not presenting a face of at least five

308

uninterrupted hydrophobic residues in the α-helix 36.

309 310

Discussion

311

In this study, we aimed at finding a short 17-mer fragment of MSI-78, while maintaining MSI-78

312

antimicrobial activity. We found two 17-mer fragments that maintained high spectrum of

313

activity, MSI-78(1-17) and MSI-78(4-20). As MSI-78(4-20) was as effective as MSI-78 against

314

all strains tested, we chose this peptide to study in more detail. Several physicochemical

315

parameters have been associated to a good AMP performance, among them: size, sequence,

316

charge, secondary structure, hydrophobicity and amphipathicity. Although MSI-78(4-20) is

317

shorter and possesses a net charge lower than MSI-78, hydrophobicity and amphipathicity

318

parameters favor MSI-78(4-20) antimicrobial activity. On the other hand, while MSI-78(4-20)

319

structures as an α-helix, the percentage of helicity is about half when comparing to MSI-78,

320

however, this does not seem to be determinant for antimicrobial activity. It has been shown that

321

AMP secondary structure and biological activity are not always coupled

322

Glycine in the N-terminus of MSI-78(4-20) might contribute to the smaller α-helical content in

323

comparison to MSI-78 as the Glycine N-capping stabilizing effect is well known

324

biophysical studies show that both peptides interact preferentially with the bacterial cell

325

membrane model, being the membrane disruption concentration-dependent

326

interaction of both peptides with the bacterial membrane initiates with peptide binding to the

327

membrane surface through electrostatic interactions, which is specially pronounced in the case of

328

MSI-78. In the case of MSI-78(4-20), its higher hydrophobicity (higher H and µH) can favour a

37, 38

. The absence of a

40

39

. The

. The proposed


15


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

Page 16 of 27

329

deeper insertion within the bacterial lipid bilayer, which may explain its ability to exert a

330

bacterial effect despite its lower ability to form α-helices

331

published MSI-78 mechanism of interaction with bacterial membrane models, involving

332

induction of substancial changes in lipid bilayers via positive curvature strain and toroidal pores

333

formation 42.

334

We have also found that MSI-78(4-20) is less hemolytic than MSI-78, which represents a major

335

advantage of the 17-mer AMP as compared to the lead MSI-78. In fact, as MSI-78(4-20)

336

displays a lower net charge than MSI-78 (+7 and +9 respectively), this can be determinant for the

337

initial interaction between the peptide and the mammalian membrane. While hydrophobicity

338

parameter values would point to a higher hemolytic activity of MSI-78(4-20), this did not occur.

339

It has been reported that increase of H and µH substantially enhance hemolytic activity, however,

340

MSI-78(4-20) does not possess a well-defined hydrophobic face, as predicted by Heliquest. For

341

MSI-78, such hydrophobic face is predicted, which may maximize the hydrophobic interactions

342

of the non-polar face of the amphipathic-helix and the lipid, in case of α-helix formation upon

343

interaction with RBC

344

substitution in the non-polar sector of a helical wheel projection has been observed to improve

345

peptide selectivity, while reduction of helical content has been associated to decreased hemolytic

346

activity 46, 47.

347

Therefore, the more pronounced hemolytic effect of MSI-78 may be attributed to the marked

348

non-specific hydrophobic interactions between the peptide and the mammalian cell membranes.

349

The perturbation of the lipid bilayers by a peptide is indeed governed by a balance between

350

electrostatic and hydrophobic interactions. Our results clearly indicate the importance of the

351

balance of these forces, suggesting this effect to be highly responsible for the MSI-78(4-20)

36, 43-45

41

. These results are in line with the

. In fact, disruption of the hydrophobic face with a lysine


16

Page 17 of 27

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352

improved selectivity towards bacterial cells and the ability to overcome the hemolytic toxic

353

effects of MSI-78. Still, differences between the peptides regarding interactions with the

354

mammalian membrane model DMPC LUVs were not observed, however a membrane model

355

does not mimic a mammalian cell with its inherent complexity. In fact to obtain the full picture

356

of the peptides mechanism of action, more detailed studies will need to be carried out, including

357

the use of more complex membrane models with other compounds present in biological

358

membranes (e.g., cholesterol, phosphatidylethanolamines, proteins), and also intact cells

359

Nevertheless, the biophysical properties reported in this study will be useful in designing

360

membrane specific antimicrobial peptides.

361

In this study, we found a very effective and broad-spectrum AMP, MSI-78(4-20). This MSI-78

362

derivative is as effective as the lead peptide, while having reduced hemolytic activity, thus

363

representing an improved version of MSI-78. We can envisage a wide application for this AMP

364

in the field of antimicrobial research and possibly in the field of anticancer therapy, as a number

365

of other synthetic Magainin analogues demonstrated promising anticancer activity 49.

48

.

366 367 368 369 370

FIGURES


17


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

Page 18 of 27

371 372

Figure 1. Hemolytic activity of MSI-78 and MSI-78(4-20) on human RBC. Peptide

373

concentrations tested covered a range from 512 µg/mL to 1 µg/mL. Untreated bacterial cells

374

were used as negative control and bacterial cells treated with 0.2% Triton X-100 were used as

375

positive control.

376

377 378

Figure 2. Zeta potential measurements of 200 µM DMPC (A) and POPC:POPG molar ratio 1:1

379

mixture (B) after 30 min incubation and stabilization with MSI-78(4-20) and MSI-78 at 37°C.

380

Circles MSI-78, squares MSI-78(4-20). Error bars represent the standard deviation of at least

381

three independent experiments.


18

Page 19 of 27

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382 383

Figure 3. Hydrodynamic diameters of 200 µM DMPC (A) and POPC:POPG 1:1 molar ratio

384

mixture (B) after 30 min incubation and stabilization with MSI-78 and MSI-78(4-20) at 37 °C.

385

Bars represent diameters (nm) and dots represent polydispersity. Error bars represent the

386

standard deviation of at least three independent experiments.

387


19


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

Page 20 of 27

388

Figure 4. CD spectra of peptides MSI-78 (solid line) and MSI-78(4-20) (dotted line) acquired in

389

aqueous buffer (A) and in the presence of DMPC (B) and POPC/POPG (C) LUVs. Heliquest

390

helical wheel projection diagrams of MSI-78 and MSI-78(4-20) (D), apolar residues are

391

represented in yellow and polar residues in blue.

392 393

TABLES

394

Table 1 – Amino acid sequences of synthetic peptides derived from MSI-78. The 17-mer

395

peptides were designed to span MSI-78 by 1-residue shift. Peptides Amino acid sequence GIGKFLKKAKKFGKAFVKILKK 22 AA MSI-78 17 AA MSI-78(1-17) GIGKFLKKAKKFGKAFV IGKFLKKAKKFGKAFVK MSI-78 (2-18) GKFLKKAKKFGKAFVKI MSI-78 (3-19) KFLKKAKKFGKAFVKIL MSI-78 (4-20) FLKKAKKFGKAFVKILK MSI-78 (5-21) LKKAKKFGKAFVKILKK MSI-78 (6-22)

396 397

Table 2 – Determination of MIC and MBC of MSI-78 and 17-mer derivatives against selected

398

microorganisms.

Peptides

399

MSI-78 MSI-78(1-17) MSI-78(2-18) MSI-78(3-19) MSI-78(4-20) MSI-78(5-21) MSI-78(6-22)

S. aureus (MRSA) MIC MBC

Microorganisms S. aureus S. epidermidis MIC MBC MIC MBC

P.aeruginosa MIC MBC

16-32 (6.5-13)

32

8-16 (3.2-6.5)

8-16

0.5-1 (0.2-0.4)

0.5-1

0.5-1 (0.2-0.4)

1-4

64-128

256

32

32-64

4-8

8-16

0.5-2

2-4

>512

-

>512

-

16-32

32-64

4-8

>16

512

>512

256

256-512

8-16

16-32

1-2

2-4

32 (16)

64

8-16 (4-8)

16-32

2 (1)

2-4

0.25-1 (0.1-0.5)

1-4

256

256-512

64-128

128-256

2-4

4

1-4

4-8

>512

-

>512

-

8-32

16-64

2-4

2-8

MIC values are presented in µg/ml. Concentrations in µM are in parenthesis.

400 401

Table 3 – Physicochemical properties and structural analysis of MSI-78 and MSI-78(4-20). Peptides

Molecular weight

Charge

H

µH

Aliphatic Index

Predicted hydrophobic face*

Helicity (% helix)

MSI-78 MSI-78(4-20)

2476 1995

+9 +7

0.241 0.322

0.674 0.704

93.18 97.65

FFFAILGLV None

62% 33%


20

Page 21 of 27

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402 403 404 405 406

The values for net charge (z), hydrophobicity (H) and mean hydrophobic moment (μH) were assessed using the analysis module of Heliquest 36. Aliphatic index was obtained using The Collection of Antimicrobial Peptides (CAMP). * Hydrophobic face predicted by Heliquest, this algorithm detects the existence of an uninterrupted hydrophobic face of at least 5 residues adjacent on a helical wheel.

407

AUTHOR INFORMATION

408

Corresponding author

409

M. Cristina L. Martins

410

INEB - Instituto de Engenharia Biomédica

411

Rua do Campo Alegre 823, 4150-180 Porto, Portugal

412

Tel: +351 22 6074982

413

Fax: +351 22 6094567

414

e-mail: [email protected]

415 416

Author Contributions

417

The manuscript was written through contributions of all authors. All authors have given approval

418

to the final version of the manuscript. ‡These authors contributed equally.

419 420

Funding Sources

421

This work was financed by FEDER funds through the Programa Operacional Factores de

422

Competitividade (COMPETE) and by Portuguese funds through FCT (Fundação para a Ciência e

423

a

424

C/SAU/LA0002/2013, Portugal-China joint Innovation Centre for Advanced Materials

Tecnologia)

in

the

framework

of

the

projects

PTDC/CTM/101484/2008,

PEst-


21


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

and

the

grants:

SFRH/BPD/79439/2011

Page 22 of 27

425

(JICAM2013)

(Claudia

Monteiro),

426

SFRH/BPD/99124/2013 (Marina Pinheiro), and SFRH/BD/89001/2012 (Catarina Seabra).

427 428

ACKNOWLEDGMENT

429

This work was financed by FEDER funds through the Programa Operacional Factores de

430

Competitividade (COMPETE) and by Portuguese funds through FCT (Fundação para a Ciência

431

e a Tecnologia) in the framework of the projects PTDC/CTM/101484/2008, PEst-

432

C/SAU/LA0002/2013, Portugal-China joint Innovation Centre for Advanced Materials

433

(JICAM2013)

434

SFRH/BPD/99124/2013 (Marina Pinheiro), and SFRH/BD/89001/2012 (Catarina Seabra).

435

The authors would like to thank to Centro Hospitalar de São João, EPE, Porto, for the human

436

buffy coats.

and

the

grants:

SFRH/BPD/79439/2011

(Claudia

Monteiro),

437 438

SUPPORTING INFORMATION AVAILABLE

439

This information is available free of charge via the Internet at http://pubs.acs.org/.

440 441

ABBREVIATIONS

442

AMP, Antimicrobial peptide; MIC, Minimum inhibitory concentration; MBC, Minimum

443

bactericidal concentration.

444 445

REFERENCES

446 447

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22

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538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 577 578 579


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580 581


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Manuscript Title: A 17-mer membrane-active MSI-78 derivative with improved selectivity

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towards bacterial cells

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Authors: Claudia Monteiro1,2‡, Marina Pinheiro3‡, Mariana Fernandes1,2, Sílvia Maia4, Catarina

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L. Seabra1,2,5,6, Frederico Ferreira-da-Silva1,7, Salette Reis3, Paula Gomes4, M. Cristina L.

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Martins1,2,5

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A 17-mer Membrane-Active MSI-78 Derivative with Improved

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