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International Journal of Antimicrobial Agents 37 (2011) 432–437

Contents lists available at ScienceDirect

International Journal of Antimicrobial Agents

journa l homepage: ht tp : / /www.e lsev ier .com/ locate / i jant imicag

nalogues of peptide SMAP-29 with comparable antimicrobial potency andeduced cytotoxicity

aymond M. Dawson ∗, Chun-Qiang LiuSTO Melbourne, 506 Lorimer Street, Fishermans Bend, VIC 3207, Australia

r t i c l e i n f o

rticle history:eceived 1 December 2010ccepted 7 January 2011

eywords:eptide

a b s t r a c t

SMAP-29 (sheep myeloid antimicrobial peptide-29) is a peptide with potent antibacterial properties.However, it is also highly cytotoxic both to human red blood cells (hRBCs) and human embryonic kidney(HEK) cells. In this study, some of the amino acids of SMAP-29 were changed in an attempt to reducehaemolytic activity whilst maintaining high antibacterial efficacy. These analogues, plus other analoguesdescribed in the literature with potent antimicrobial activity against Gram-positive bacteria coupled

MAP-29ytotoxicityaemolysisherapeutic index

with no or low haemolytic activity, were evaluated for their cytotoxicity (hRBCs and HEK cells) as well asantimicrobial efficacy against two Gram-positive (Bacillus anthracis and Bacillus globigii) and two Gram-negative bacteria (Escherichia coli and Burkholderia thailandensis). The analogues previously described inthe literature were found to have low antibacterial and haemolytic activity. Two of the designed analogueshad comparable antibacterial efficacy with SMAP-29 against B. anthracis but reduced haemolytic activityand therefore had a therapeutic index that was enhanced 2.3–2.6-fold over that of SMAP-29.

Publ

Crown Copyright © 2011

. Introduction

SMAP-29 (sheep myeloid antimicrobial peptide-29) is a potentntimicrobial peptide with a minimum inhibitory concentrationMIC) against Bacillus anthracis (Sterne strain vaccine) of 1.5 �M1]. It is also highly haemolytic (human erythrocytes) and cyto-oxic [human embryonic kidney (HEK) cells] at concentrationsf 6–25 �M [1]. A number of variants of this peptide have beenrepared in an attempt to improve the selectivity for pathogenicicroorganisms over mammalian cells [2]. We have obtained some

f the more promising analogues for evaluation of their antimicro-ial and cytotoxic properties, in conjunction with some analogueshat we have designed ourselves. The results are reported below.

. Materials and methods

.1. Bacterial strains and growth media

The Sterne strain 34F2 of B. anthracis was purchased from Fort

odge Australia Pty Ltd. (Colorado Serum Co., Denver, CO). This

train is not pathogenic to humans because it lacks the capsule ofirulent strains [3]. However, there is evidence that absence or pres-nce of the capsule has no effect on the antimicrobial potency of

∗ Corresponding author. Tel.: +61 3 9626 8477; fax: +61 3 9626 8410.E-mail address: ray.dawson@dsto.defence.gov.au (R.M. Dawson).

924-8579/$ – see front matter. Crown Copyright © 2011 Published by Elsevier B.V. on beoi:10.1016/j.ijantimicag.2011.01.007

ished by Elsevier B.V. on behalf of International Society of Chemotherapy. All rights reserved.

peptides [4]. Bacillus subtilis var. niger ATCC 9372 (Bacillus globigii)is a non-pathogenic simulant for B. anthracis [5]. Burkholderia thai-landensis, often used as a surrogate for Burkholderia pseudomallei,the causative agent of melioidosis [6], was kindly provided by Prof.I. Beacham (Griffith University, Brisbane, Australia). Escherichia coliK91BK was kindly provided by Prof. G. Smith (Missouri Univer-sity, Columbia, MO). NZCYM growth medium was obtained fromSigma-Aldrich (Sydney, Australia).

2.2. Human cells

Human red blood cells (hRBCs) were obtained free of chargeunder a contract with the Australian Red Cross Blood Service (Mel-bourne, Australia). HEK 293s cell line CRL-1573.3 was obtainedfrom the American Type Culture Collection (ATCC, Manassas, VA).

2.3. Peptides and other materials

SMAP-29 and its analogues (73–96% purity) were synthesisedwith an N-terminal acetate and C-terminal amide by Beijing SBSGenetech (Beijing, China). Solutions of 2 mM SMAP-29 in sterilewater were stored at −70 ◦C in 40 �L aliquots until needed. All otherreagents were obtained from Sigma-Aldrich.

2.4. Antibacterial assays

A 5 mL aliquot of sterile NZCYM growth medium was inocu-lated with a colony of test bacteria and was incubated overnight

half of International Society of Chemotherapy. All rights reserved.

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R.M. Dawson, C.-Q. Liu / International Jour

t 37 ◦C with shaking at 200 rpm. A 1 mL aliquot was diluted00-fold with fresh medium and was incubated for a further 1 hith shaking at 37 ◦C. Meanwhile, 400 �L aliquots of diluted pep-

ides in growth medium were placed in selected wells of a sterileolystyrene 48-well cell culture plate (cat. no. 3548; Corning Costar,cton, MA), which had been treated with 1 mL of 0.5% bovineerum albumin (BSA) in phosphate-buffered saline (PBS) to blockeptide-binding sites [7], and were washed twice with 1 mL ofBS. These aliquots were serially diluted (200 �L + 200 �L) alonghe plate. Aliquots (200 �L) of the bacterial suspension were thendded to the 200 �L aliquots of peptide solution. Absorbance at00 nm (A600) of the peptide–bacteria mixture was read in a BioTekynergyTM HT plate reader (BioTek Instruments, Winooski, VT)nd the plate was incubated at 30 ◦C with shaking at 150 rpmor 8 h. Further readings of A600 were taken at hourly intervals.ontrol wells contained no peptide (positive control) or no bac-eria (blank). No growth of bacteria was observed in antibioticontrol wells at concentrations of >25 �g/mL tetracycline (56 �M)or E. coli, 60 �g/mL (135 �M) tetracycline for B. thailandensis and�g/mL (0.3 �M) vancomycin for B. globigii and B. anthracis. TheIC, defined as the lowest concentration of peptide that completely

revented growth of the bacteria over 8 h, and the I50 [concentra-ion of peptide that reduced the net absorbance (assay − blank)y 50%] were determined. All experiments were repeated at

east once.To determine the minimum bactericidal concentration (MBC)

or selected peptides versus B. anthracis, 20 �L aliquots from wellshowing no growth were streaked onto nutrient agar plates, whichere incubated for 16 h at 37 ◦C. The MBC was determined as the

owest concentration of peptide in the initial assay for which theumber of colonies of bacteria observed on the agar plate was <0.1%f the colonies in control wells [8].

.5. Haemolysis

Packed RBCs (2 mL) were suspended in 20 mL of cold PBS,ashed with 3 × 20 mL of cold PBS in a Sigma 3-18K centrifuge at◦C, 1000 × g for 10 min, and re-suspended in 100 mL of cold PBS.hen, 20 �L aliquots of 2 mM peptides were diluted to 200 �L withBS in the wells of a Nunc-ImmunoTM MicroWellTM 96-well flat-ottom plate (cat. no. 43954; Nunc A/S, Roskilde, Denmark) anderially diluted (100 �L + 100 �L) along the plate. Dilute erythro-ytes (100 �L) were added to each 100 �L aliquot of peptide andhe plate was incubated for 30 min at 37 ◦C at 100 rpm. The plateas centrifuged at 1000 × g for 20 min in a Heraeus® multifuge®

S-R centrifuge (DJB Labcare Ltd., Newport Pagnell, UK). Aliquots100 �L) of the well supernatants were transferred to a fresh 96-ell plate and the absorbance at 414 nm (A414) was read in the plate

eader. Controls (in triplicate) were 0.1% Triton X-100 in PBS (pos-tive control) and PBS only (blank). Each experiment was repeatedt least once on separate days for each of two to four separate sam-les of washed blood cells. Most assays were completed within 10ays of receipt of the packed RBCs; occasionally, however, repli-ate assays were performed 24–37 days after receipt. No trend forncreased fragility of the RBCs (increased haemolysis) with lengthf time in storage (at 2–4 ◦C) was observed. The maximum con-entration of peptide that caused ≤1% haemolysis (MHC) and theoncentration of peptide that caused 50% haemolysis (H50) wereetermined.

.6. Cytotoxicity

HEK 293s cells were grown in Advanced DMEM/F12 (Invitrogen,ulgrave, VIC, Australia) supplemented with 2 mM l-glutamine

nd 2% (w/v) heat-inactivated foetal bovine serum at 37 ◦C, 5% CO2o reach 60–80% confluence, treated with 0.05% trypsin/ethylene

Antimicrobial Agents 37 (2011) 432–437 433

diamine tetra-acetic acid (EDTA) and re-suspended in the samegrowth medium to a final concentration of ca. 2 × 105 cells/mL. A200 �L aliquot of the cell suspension was seeded per well in a Corn-ing Costar 48-well flat-bottom microtitre plate (Sigma-Aldrich) andincubated overnight (ca. 16 h) at 37 ◦C in an atmosphere contain-ing 5% CO2. Antimicrobial peptides were diluted in 50 �L of freshmedium and were added to each well to final concentrations of 0,0.78, 1.56, 3.13, 6.25, 12.5, 25, 50 or 100 �M. The plate was incu-bated at 37 ◦C in 5% CO2 for 4 h. An aliquot (25 �L) of alamarBlue®

reagent (Invitrogen) was added to each well and the plate wasread immediately at 530 nm (excitation) and 590 nm (emission).The plate was returned to the 37 ◦C/5% CO2 incubator for a further2 h and was read again at 530/590 nm. The fluorescence intensity inthe well reflects the extent of metabolic activity of the cells, whichis assumed to be directly proportional to the cell number [9]. HEKcells without peptides were used as positive controls. Completekilling of cells in the well was defined as no change in fluorescenceintensity in the presence of alamarBlue® reagent over the 2 h incu-bation at 37 ◦C in 5% CO2. The concentration of peptide that reducedthe net fluorescence (positive control − total cell death control) by50% (C50) was determined.

3. Results

3.1. Design and selection of SMAP-29 analogues

The C-terminal amino acid of SMAP-29 (glycine) is thought to bepost-translationally modified into a NH2 group to form a peptideof 28 amino acids with a C-terminal amide, known as SMAP-28,but sometimes called SMAP-29 in the literature (a source of confu-sion) [2]. In this paper, SMAP-29 refers to the peptide of 29 aminoacids, and SMAP-28 to the peptide of 28 amino acids. The antimi-crobial activity of SMAP-29 has been attributed to the N-terminalamphipathic �-helix, and the haemolytic/cytotoxic activity to thehydrophobic C-terminal region [10]. In an attempt to reducethe haemolytic activity, four SMAP-29 analogues were designed,namely SMAP-29 (T27,29), SMAP-29 (G1A2T11A13), SMAP-29 (K8)and SMAP-29 (G17). First, the hydrophilic amino acid threonine wasintroduced at each of positions 27 and 29 in place of the hydropho-bic amino acid isoleucine and the amphiphilic amino acid glycine,respectively, forming SMAP-29 (T27,29). The design of the secondand third SMAP-29 analogues above was based on previous studiesby Tossi et al. [11,12] who analysed the sequences of a databasecomprising the naturally occurring amphipathic �-helical antimi-crobial peptides (of which SMAP-29 is one) and identified somecommon themes. A template for the sequences of the first 18 aminoacids is shown in Fig. 1.

Glycine occurred at position 1 in >70% of the sequences anal-ysed, and lysine occurred at position 8 in >50% of cases. For thefirst 18 amino acids of SMAP-29, 14 of the 18 fit the above tem-plate (whilst noting that 3 positions can be any amino acid). Thepositions of misfit are 1 (arginine instead of glycine), 2 and 13(the neutral amphiphilic glycine instead of a hydrophobic residue)and 11 (hydrophobic alanine instead of the hydrophilic cationic oranionic residues that predominate in the database). Accordingly,a SMAP-29 analogue, named SMAP-29 (G1A2T11A13), was synthe-sised in which the arginine at position 1 was changed to glycine,the glycine at position 2 to alanine (slightly more hydrophobic),the alanine at position 11 to threonine (mildly hydrophilic) andthe glycine at position 13 to alanine. SMAP-29 (K8), with lysine

at position 8 instead of arginine, was also synthesised in accor-dance with the preponderance of lysine at position 8 in the peptidesof the database. Finally, SMAP-29 (G17), with glycine in place oftyrosine at position 17, was synthesised because of the observa-tion that haemolytic activity is directly correlated with the angle

434 R.M. Dawson, C.-Q. Liu / International Journal of Antimicrobial Agents 37 (2011) 432–437

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ubtended by the hydrophobic sector of the �-helix, and that anninterrupted sector of five hydrophobic residues (angle of 120◦)

s sufficient for good antimicrobial activity with reduced haemoly-is [13]. This angle is 140◦ for the hydrophobic sector of positions–17 of SMAP-29, which constitute its �-helix [14]. Reducing theydrophobic sector of the �-helix of SMAP-29 slightly may there-

ore improve its bacterial cell–erythrocyte selectivity. Replacementf the hydrophobic tyrosine at position 17 with the amphiphiliclycine should achieve this objective. This substitution is contraryo the template in Fig. 1 that specifies that position 17 should beydrophobic, although glycine borders on being hydrophobic (A.ossi, pers. comm.). SMAP-29 linked via the C-terminus to pep--K, a variant of the cell-penetrating peptide pep-1 [15] (hereamed SMAP-Pep) as well as SMAP-29 conjugated to BSA via the-terminus and a linker sequence (GGGGSC) (named SMAP-BSA),ere also prepared for evaluation.

Two peptides derived from SMAP-28 and SMAP-29 by delet-ng 11 amino acids from the C-terminal end [SMAP-28 (1–17) andMAP-29 (1–18), respectively] have also been reported by othersnd their antimicrobial and haemolytic properties studied [2]. TheIC of SMAP-28 (1–17) was found to be in the low micromolar

ange against Gram-positive bacteria, yet it showed no haemoly-is at 100 �M. It was somewhat less active against Gram-negativeacteria [2]. SMAP-29 (1–18), on the other hand, has MICs as lows 0.3–0.6 �M against two Gram-negative bacteria, but in excessf 10 �M against Gram-positive bacteria. It is also relatively non-aemolytic (4% haemolysis at 50 �M) [2]. We chose to evaluateoth these truncated versions of SMAP-29 against two Gram-egative and two Gram-positive bacteria (see Section 2.1), as well

s SMAP-28 (1–17, K2,7,13), SMAP-28 (K22,25,27) [2] and a relatedeptide, novispirin G-10 [16], all of which have been reported toave MICs in the low micromolar range against Gram-positive andram-negative bacteria coupled with no or low haemolytic activ-

able 1mino acid sequences of SMAP-29 and its variants.

Peptide Sequencea

SMAP-29 RGLRRLGRKIAHGVKKYGPTVLRIIRIAGSMAP-28 (1–17) RGLRRLGRKIAHGVKKYSMAP-29 (1–18) RGLRRLGRKIAHGVKKYGSMAP-28 (K22,25,27) RGLRRLGRKIAHGVKKYGPTVKRIKRKA-NH2

SMAP-28 (1–17, K2,7,13) RKLRRLKRKIAHKVKKYNovispirin G-10 KNLRR I IRKGIH I I KKYGSMAP-29 (T27,29) RGLRRLGRKIAHGVKKYGPTVLRIIRTATSMAP-29 (K8) RGLRRLGKKIAHGVKKYGPTVLRIIRIAGSMAP-29 (G1A2T11A13) GALRRLGRKITHAVKKYGPTVLRIIRIAGSMAP-29 (G17) RGLRRLGRKIAHGVKKGGPTVLRIIRIAGSMAP-BSA RGLRRLGRKIAHGVKKYGPTVLRIIRIAGGGGGSC-

(BSA)SMAP-Pep RGLRRLGRKIAHGVKKYGPTVLRIIRIAG-

KKTWWKTWWTKWSQPKKKRKVPep-1-K KKTWWKTWWTKWSQPKKKRKV

a All peptides were synthesised with an N-terminal acetate and C-terminal amide.

y occurring amphipathic �-helical antimicrobial peptides [11,12].

ity [2]. The sequences of all the peptides mentioned above, newand already reported, are listed in Table 1. They were all synthe-sised with an N-terminal acetate and C-terminal amide, as this is aform in which they would be encountered in fusion peptides, whichis another strategy to increase selectivity. C-terminal modificationalso often enhances antimicrobial activity [17].

3.2. Antibacterial activity

The antibacterial activities of SMAP-29 and its analoguestowards Gram-positive bacteria (B. globigii and B. anthracis) aredocumented in Table 2. None of the published SMAP-29 vari-ants tested (the first five entries in Table 2 after SMAP-29 itself)had MICs <10 �M against B. globigii, in contrast to their reportedbehaviour [with the exception of SMAP-29 (1–18)] against two tofour other Gram-positive bacteria (see references in [2]). They wereall inactive against B. anthracis. On the other hand, the four SMAP-29 analogues that we designed and synthesised, plus SMAP-BSA,SMAP-Pep and pep-1-K itself, all had MICs <10 �M against B. glo-bigii, and five of the seven peptides had MICs ≤10 �M against B.anthracis, the exceptions being pep-1-K (MIC = 53 �M) and SMAP-29 (G17) (MIC = 70 �M). None was significantly more potent thanSMAP-29 itself against either bacterium, although the antibacterialactivity was comparable in most cases. The MBC for the peptidesagainst B. anthracis was 1.2–2.5× the MIC (Table 2).

The antibacterial activities of SMAP-29 and its analoguestowards Gram-negative bacteria (E. coli and B. thailandensis) aregiven in Table 3. Again, the reported activity of the five knownSMAP-29 analogues against Gram-negative bacteria (see referencesin [2]) could not be reproduced and all five peptides were inactive.The MICs of our six analogues against E. coli ranged from 20 �Mto 100 �M (22 �M for SMAP-29 itself), but the only peptide toshow any activity against B. thailandensis, apart from SMAP-29 itself(MIC = 71 �M), was SMAP-29 (G1A2T11A13) (MIC = 84 �M). Theseresults are not surprising in that Burkholderia spp. are intrinsicallyresistant to a wide range of antimicrobial agents [18].

3.3. Haemolytic activity and cytotoxicity

The extent of haemolysis of hRBCs induced by the peptidesis shown in Fig. 2, where the amount of non-haemolysed bloodcells (percentage of control) is plotted against peptide concentra-tion. Fig. 2a shows the results for peptides where little haemolysisoccurred [the five known SMAP-29 analogues, SMAP-29 (G17) andpep-1-K] and Fig. 2b shows the results for the rest (SMAP-29 itselfand five of our six designed analogues). Note that the y-axis scaleis 90–100% for Fig. 2a and 0–100% for Fig. 2b. There was consid-

erable variability for replicate assays on different days from thesame blood sample (unrelated to the time in storage of the RBCssince receipt from the Red Cross), and sometimes even more vari-ation between mean values of haemolysis from different donors.Consequently, the error bars in Fig. 2 are relatively large. The low

R.M. Dawson, C.-Q. Liu / International Journal of Antimicrobial Agents 37 (2011) 432–437 435

Table 2Antibacterial activity of SMAP-29 analogues against Gram-positive bacteriaa.

Peptide Bacillus globigii Bacillus anthracis

MICb I50c MIC I50 MBCd

SMAP-29 1.37 ± 0.09 (11) 0.46 ± 0.04 (8) 1.38 ± 0.03 (17) 0.90 ± 0.04 (17) 1.79 ± 0.11 (5)SMAP-28 (1–17) 59.5 ± 4.5 (4) 39.7 ± 5.3 (3) >100 (3) >100 (3)SMAP-29 (1–18) 50.0 ± 4.8 (5) 35.4 ± 0 (5) >100 (3) >100 (3)SMAP-28 (K22,25,27) 12.5 ± 0 (4) 4.42 ± 0.27 (4) >100 (4) >100 (4)SMAP-28 (1–17, K2,7,13) 57.4 ± 6.5 (5) 23.3 ± 2.6 (5) >100 (2) >100 (2)Novispirin G-10 12.5 ± 1.2 (5) 2.78 ± 0.28 (3) >100 (4) >100 (4)SMAP-29 (T27,29) 3.13 ± 0 (4) 1.01 ± 0.07 (4) 2.51 ± 0.14 (7) 1.48 ± 0.08 (7) 6.25 ± 0 (2)SMAP-29 (K8) 1.86 ± 0.14 (4) 0.65 ± 0.05 (4) 1.38 ± 0 (6) 0.90 ± 0 (6) 3.13 ± 0 (3)SMAP-29 (G1A2T11A13) 1.31 ± 0.10 (8) 0.52 ± 0.05 (6) 1.19 ± 0.06 (14) 0.80 ± 0.04 (14) 1.97 ± 0.20 (3)SMAP-29 (G17) 7.87 ± 0.79 (3) 1.10 ± 0 (2) 70.2 ± 14.1 (3) 40.8 ± 15.5 (3)SMAP-BSA 6.25 ± 0 (2) 4.42 ± 0 (2) 10.1 ± 0.4 (5) 6.17 ± 0.43 (5) 12.5 ± 0 (3)SMAP-Pep 0.93 ± 0.13 (4) 0.55 ± 0.18 (4) 3.49 ± 0 (3) 2.28 ± 0 (3) 4.42 ± 0.66 (2)Pep-1-K 3.94 ± 0.39 (3) 1.24 ± 0.16 (3) 52.6 ± 10.0 (4) 22.3 ± 2.2 (4)

a Data are given as geometric mean ± standard error of the mean in units of �M, with the number of experiments in parentheses. MIC, I50 and MBC were measured after8 h of incubation of the peptides with bacteria at 30 ◦C.

b MIC (minimum inhibitory concentration) is the lowest concentration of peptide that completely inhibits growth of the bacteria, as reflected by the absorbance at 600 nm.c I50 is the concentration that reduces the net absorbance (control − blank) at 600 nm by 50%.d MBC (minimum bactericidal concentration) is the lowest concentration of peptide that reduces number of colonies of bacteria to <0.1% of control, as determined by

streaking aliquots onto nutrient agar plates.

Table 3Antibacterial activity of SMAP-29 analogues against Gram-negative bacteriaa.

Peptide Escherichia coli Burkholderia thailandensis

MICb I50c MIC I50

SMAP-29 22.3 ± 1.1 (6) 15.2 ± 1.4 (7) 70.7 ± 10.6 (4) 50.0 ± 6.7 (6)SMAP-28 (1–17) ≥100 (4) ≥100 (4) >100 (2) >100 (2)SMAP-29 (1–18) >100 (2) >100 (2) >100 (2) >100 (2)SMAP-28 (K22,25,27) >100 (2) >100 (2) >100 (2) >100 (2)SMAP-28 (1–17, K2,7,13) >100 (2) >100 (2) >100 (2) >100 (2)Novispirin G-10 ≥100 (3) 100 ± 15 (3) >100 (2) >100 (2)SMAP-29 (T27,29) 36.1 ± 3.2 (3) 20.3 ± 0.9 (3) >100 (2) ≥100 (2)SMAP-29 (K8) 31.5 ± 3.2 (3) 15.8 ± 0.8 (3) >100 (2) 84.1 ± 6.3 (2)SMAP-29 (G1A2T11A13) 28.7 ± 1.7 (5) 14.4 ± 0.9 (5) 84.1 ± 15.9 (4) 42.0 ± 5.5 (4)SMAP-29 (G17) 100 ± 0 (2) 59.5 ± 4.5 (2) >100 (2) >100 (2)SMAP-BSA 100 (1) 50 (1) >100 (1) >100 (1)SMAP-Pep 19.8 ± 2.0 (3) 8.84 ± 0 (3) >100 (1) >100 (1)Pep-1-K >100 (2) >100 (2) >100 (2) >100 (2)

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that cnm b

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a Data are given as the geometric mean ± standard error of the mean in units of �f incubation of the peptides with bacteria at 30 ◦C.b MIC (minimum inhibitory concentration) is the lowest concentration of peptidec I50 is the concentration that reduces the net absorbance (control − blank) at 600

aemolytic activity of the known SMAP-29 analogues and of pep--K is consistent with the literature [2,15]. Haemolytic activityan be characterised by the maximum haemolytic concentrationMHC), i.e. the highest concentration that causes no haemolysis [19]defined as ≤1% haemolysis in our study), or the concentration that

ig. 2. Haemolytic activity of SMAP-29 and its analogues against human red blood cells.amples (i.e. two to four different donors), with two to four replicates for each sample (u

th the number of experiments in parentheses. MIC and I50 were measured after 8 h

ompletely inhibits growth of the bacteria, as reflected by the absorbance at 600 nm.y 50%.

causes 50% haemolysis (H50) [20]. For our results, the MHC is nota suitable measure of haemolysis, as shown by Fig. 2a; it is neces-sary to reduce the concentration of peptide to ≤6 �M for three ofthe peptides in this graph to cause haemolysis to fall below 1%, buthaemolysis is no more than 4% at 100 �M peptide. We therefore

Results are shown as the mean ± standard error of the mean of two to four bloodp to seven in the case of SMAP-29).

436 R.M. Dawson, C.-Q. Liu / International Journal of Antimicrobial Agents 37 (2011) 432–437

Table 4Therapeutic ratios of SMAP-29 analogues.

Peptide Haemolytic activity Cytotoxicity

H50 (�M)a H50/I50b C50

c C50/I50

SMAP-29 23.1 25.7 1.61 1.79SMAP-28 (1–17) �100SMAP-29 (1–18) �100SMAP-28 (K22,25,27) �100SMAP-28 (1–17, K2,7,13) �100Novispirin G-10 �100SMAP-29 (T27,29) 87.6 59.2 3.08 2.08SMAP-29 (K8) 60.5 67.2 3.18 3.53SMAP-29 (G1A2T11A13) 18.4 23.0 1.63 2.04SMAP-29 (G17) �100 �2.5 9.87 0.24SMAP-BSA 146 23.7 2.14 0.35SMAP-Pep 3.04 1.3 4.35 1.91Pep-1-K �100 �4.5 18.6 0.83

measd

nm bells a

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apcfppItC

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•

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a H50 is the concentration that causes 50% haemolysis of human red blood cellsetermined from Fig. 2.b I50 is the concentration that reduces the net absorbance (control − blank) at 600c C50 is the concentration that kills 50% of human embryonic kidney (HEK) 293s c

etermined the values of H50 from the data of Fig. 2b. Either atraight line or a second-order polynomial could be well fitted tohe data when it was transformed to a linear–log relationship.

Cytotoxicity was determined only for those peptides with highntimicrobial activity (MIC ≤ 10 �M) against one or both Gram-ositive bacteria (Table 2) in order to conserve materials. Theytotoxicity results (Fig. 3) were analysed in the same way as thoseor haemolysis to determine values of C50, the concentration ofeptide that kills 50% of HEK 293s cells in 4 h (Table 4). A thera-eutic index for each peptide was calculated as the ratio of H50 to

50 (haemolysis index) or C50/I50 (cytotoxicity index) (where I50 ishe I50 of the peptide against B. anthracis). The values of H50/I50 and50/I50 are also given in Table 4.

An analysis of Table 4 indicates the following points.

Peptides that have little or no antibacterial activity against B.anthracis (Table 2) and two Gram-negative bacteria (Table 3) have

negligible haemolytic activity.SMAP-Pep is extremely haemolytic (H50 = 3 �M; MHC < 0.05 �M)and is 6–8 times as haemolytic as the two next most potentpeptides, SMAP-29 (G1A2T11A13) and SMAP-29. Consequently,

100

80

SMAP-29

SMAP-29 (T27,29

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60

SMAP-29 (K8 )

SMAP-29 (G1A

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Peptide concentration (μM)

ig. 3. Cytotoxicity of SMAP-29 and its analogues to human embryonic kidney (HEK)93s cells. Results are shown as the mean ± standard error of the mean of triplicatessays.

ured after 30 min of incubation of dilute red blood cells with peptide at 37 ◦C, as

y 50% for Bacillus anthracis from Table 2.fter 2 h at 37 ◦C as determined from Fig. 3.

SMAP-Pep has a therapeutic index (haemolysis) of only 1.3 com-pared with 25.7 for SMAP-29.

• Compared with SMAP-29, the therapeutic index (haemolysis) isessentially unchanged for SMAP-29 (G1A2T11A13) and SMAP-BSA.

• Modification of one or two amino acids of SMAP-29 to produceSMAP-29 (K8) and SMAP-29 (T27,29), respectively, has increasedthe therapeutic index (haemolysis) by 2.6-fold and 2.3-fold,respectively, by decreasing the haemolytic activity.

• The results for cytotoxicity do not parallel those for haemoly-sis. Of the SMAP-29 analogues and other peptides, only SMAP-29(G1A2T11A13) is not less cytotoxic than SMAP-29.

• Therapeutic indexes for cytotoxicity are low (0.2–3.5). Three pep-tides [SMAP-29 (G17), SMAP-BSA and pep-1-K] have a therapeuticindex (cytotoxicity) that is <1, and less than that for SMAP-29(1.79).

• As for haemolysis, SMAP-29 (K8) has the highest therapeuticindex for cytotoxicity, twice that of SMAP-29.

4. Discussion

The reported potent antibacterial activity (MIC < 10 �M) againstGram-positive bacteria of SMAP-28 (1–17), SMAP-28 (K22,25,27),SMAP-28 (1–17, K2,7,13) and novispirin G-10 (see references in [2])could not be substantiated in the present work, even though oneof the bacteria (B. globigii, also known as B. subtilis) is common toboth studies for SMAP-28 (1–17), SMAP-28 (K22,25,27) and SMAP-28(1–17, K2,7,13). The reason for this is unclear, although a differ-ent assay procedure (radial diffusion) was used in the reportedstudy for novispirin G-10 at a higher temperature (37 ◦C) than inthe present work (30 ◦C). The antimicrobial activity of some pep-tides is sensitive to significant concentrations of NaCl, and theNaCl component of the growth media used (NZCYM) is 86 mM.However, the presence of this significant concentration of NaCldoes not explain the differences in results outlined above, because100 mM NaCl increased the MIC only two-fold relative to no NaClfor SMAP-28 (1–17), whilst causing the MIC to be unchanged ortwo-fold lower (i.e. increased potency) for SMAP-28 (K22,25,27) andSMAP-28 (1–17, K2,7,13). The temperature (37 ◦C) and growth media(Luria–Bertani broth with 1% peptone) of the literature study maybe factors in the differing results. Similarly, the reported potent

antibacterial activity against E. coli (MIC = 4–16 �M) of SMAP-28(1–17), SMAP-28 (K22,25,27) and SMAP-28 (1–17, K2,7,13) could notbe repeated; in fact, the MIC was found to be ≥100 �M in our study.This could be a function of the different strain of E. coli used, inthat a MIC of 0.07–0.86 �M was found in three separate studies for

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MAP-29 itself against this microorganism [2], in contrast to 22 �Mn the present work (Table 3). As for the Gram-positive bacteria, theifferent temperatures and growth media used may also be influ-ncing factors. The effects of physiological concentrations of NaCl140–150 mM) and serum were not studied in the reported [2] orresent work.

The antibacterial potency against the two Gram-positive Bacil-us spp. of three of the four SMAP-29 analogues that we designed

as within a factor of 2.5 of that for SMAP-29 itself (Table 2).he fourth analogue [SMAP-29 (G17)], however, although reason-bly potent against B. globigii (MIC = 7.9 �M, 5.7-fold higher thanhe MIC for SMAP-29), was relatively inactive against B. anthracisMIC = 70 �M).

Despite similar antibacterial activity, the haemolytic profilef our designed peptides differed substantially in most casesrom that of SMAP-29 (Fig. 2). SMAP-Pep was more haemolytichilst the other peptides, except SMAP-29 (G1A2T11A13), were

ess haemolytic (Fig. 2). Changing the hydrophobic C-terminal tailf SMAP-29 by replacing certain hydrophobic amino acids withhe hydrophilic lysine [SMAP-28 (K22,25,27)] or threonine [SMAP-29T27,29)] has had the predicted effect of reducing haemolytic activ-ty (Fig. 2; Table 4), but at the expense of substantially reducedntibacterial activity against B. anthracis in the case of SMAP-9 (K22,25,27) (Table 2). Blocking the C-terminal of SMAP-29 byep-1-K or BSA does not appear to affect its haemolytic activ-

ty.The H50/I50 therapeutic index (Table 4) would normally sug-

est that SMAP-29 itself (index = 26) is not sufficiently selectiveor bacterial cells to be considered for therapeutic purposes, apartrom possible topical application. Nevertheless, SMAP-29 has beenirectly inserted into lamb lungs with pneumonia, with benefi-ial therapeutic effects; the dose of SMAP-29 used was half thathich was well tolerated by healthy lambs and which did notroduce any pathology [21]. Furthermore, the closely related pep-ide SMAP-28, when injected intraperitoneally, protected micerom the lethal effects of three bacteria and protected rats fromwo models of septic shock [2]. The SMAP-29 analogues SMAP-9 (T27,29) and SMAP-29 (K8), with H50/I50 indexes of 59–67, overouble that of SMAP-29, therefore show considerable promiseor development as parenteral antimicrobial drugs. SMAP-29 (K8)lso has a two-fold higher C50/I50 therapeutic index than SMAP-9. Our results therefore show that further manipulation of themino acid structure of SMAP-29 may allow even further reduc-ions in toxicity of this peptide whilst retaining its antimicrobialctivity.

cknowledgment

The authors are grateful to the Australian Red Cross Blood Ser-ice (Melbourne, Australia) for the gift of human erythrocytes.

Funding: All funding for this research was provided by the Aus-ralian Government Department of Defence.

[

Antimicrobial Agents 37 (2011) 432–437 437

Competing interests: None declared.Ethical approval: Not required.

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