Anti-plasmodial Action of de Novo-Designed, Cationic, Lyisne Branched Amphipathic Helical Peptides

7/27/2019 Anti-plasmodial Action of de Novo-Designed, Cationic, Lyisne Branched Amphipathic Helical Peptides

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Anti-plasmodial action ofde novo-designed,cationic, lysine-branched, amphipathic,helical peptidesKaushiket al.

Kaushiket al. Malaria Journal2012, 11 :256

http://www.malariajournal.com/content/11/1/256


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1 R E S E A R C H Open Access

2 Anti-plasmodial action ofde novo-designed,3 cationic, lysine-branched, amphipathic,4 helical peptides5 Naveen K Kaushik, Jyotsna Sharma and Dinkar Sahal*

6 Abstract

7 Background: A lack of vaccine and rampant drug resistance demands new anti-malarials.

8 Methods: In vitro blood stage anti-plasmodial properties of several de novo-designed, chemically synthesized,

9 cationic, amphipathic, helical, antibiotic peptides were examined against Plasmodium falciparum using SYBR10 Green assay. Mechanistic details of anti-plasmodial action were examined by optical/fluorescence microscopy

11 and FACS analysis.

12 Results: Unlike the monomeric decapeptides {(Ac-GXRKXHKXWA-NH2) (X=F,F) (Fm, Fm IC50 >100 M)}, the

13 lysine-branched,dimeric versions showed far greater potency {IC50 (M) Fd 1.5 , Fd 1.39}. The more helical

14 and proteolytically stable Fd was studied for mechanistic details. Fq, a K-K2 dendrimer ofFm and (Fm)215 a linear dimer ofFm showed IC50 (M) of 0.25 and 2.4 respectively. The healthy/infected red cell selectivity

16 indices were >35 (Fd), >20 (Fm)2 and 10 (Fq). FITC-Fd showed rapid and selective accumulation in

17 parasitized red cells. Overlaying DAPI and FITC florescence suggested thatFd binds DNA. Trophozoites and

18 schizonts incubated with Fd (2.5 M) egressed anomalously and Band-3 immunostaining revealed them not

19 to be associated with RBC membrane. Prematurely egressed merozoites from peptide-treated cultures were

20 found to be invasion incompetent.

21 Conclusion: Good selectivity (>35), good resistance index (1.1) and low cytotoxicity indicate the promise ofFd22 against malaria.

23 Keywords:Anomalous egress, Anti-plasmodial peptides, De novopeptide design, Kinetics of peptide uptake,

24 Peptide binding to DNA, Plasmodium falciparum

25 Background26 The devastating diseases caused by protozoan para-

27 sites are a major burden of the tropics, and in par-

28 ticular, Plasmodium falciparum, the causative agent of

29 falciparum malaria, creates a serious public health prob-

30 lem in many areas of the densely populated developing

31 world. The widespread resistance of P. falciparum to32 chloroquine (CQ), which has spread from Asia to Africa,

33 has rendered the drug ineffective against the most danger-

34 ous Plasmodium strain in many affected regions of the

35 world. Unfortunately, CQ-resistance is associated with

36 cross-resistance to other quinoline drugs, such as quinine

37and amodiaquine [1]. Plasmodium falciparum is genetic-

38ally diverse and has multiple independent origins of muta-

39tions in genes that confer resistance to widely used

40anti-malarial drugs [2]. Left with just artemisinin to fight

41against malaria, Arata Kochi, Director of the Malaria

42Division at the World Health Organization, had felt com-

43pelled to say

if we lose artemisinin, we will no longer have44an effective cure for malaria[3]. However, most recently,

45alarming signs of clinical resistance against artemisinin, in

46the form of delayed parasite clearance, are being observed

47in the border between Cambodia and Thailand [4,5]. The

48challenge of designing an effective vaccine along trad-

49itional lines against malaria is that many P. falciparum

50proteins are highly polymorphic and their functions are

51redundant [6]. More than 200 million new malaria cases* Correspondence:[email protected]

Malaria Research Group, International Centre for Genetic Engineering and

Biotechnology, Aruna Asaf Ali Marg, New Delhi 110067, India

2012 Kaushik et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the CreativeCommons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, andreproduction in any medium, provided the original work is properly cited.

Kaushiket al. Malaria Journal2012,11:256

mailto:[email protected]://creativecommons.org/licenses/by/2.0http://creativecommons.org/licenses/by/2.0mailto:[email protected]


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52 reported annually is a challenge [7] that underscores the

53 urgent requirement for new drugs against malaria.

54 Peptides are an essential component of defence mech-

55 anism of all life forms and anti-microbial peptides are

56 evolutionarily ancient biological weapons. Their wide-

57 spread distribution throughout the living kingdom sug-

58 gests that anti-microbial peptides may have served a

59 fundamental role in the successful evolution of complex

60 multi-cellular organisms [8]. Despite their ancient lineage,

61 anti-microbial peptides have remained effective defensive

62 weapons, defeating the general belief that bacteria, fungi

63 and viruses can and will develop resistance to any conceiv-

64 able substance. Among other differences, uniquely anionic

65 charge on bacterial surface is a curious feature that distin-

66 guishes the prokaryotic bacteria from their eukaryotic

67 counterparts [9]. Anti-microbial peptides gain selectiv-

68 ity from their ability to target this previously under-

69 appreciated microbial Achilles heel[10-12]. Interestingly,70 a seminal feature of the malaria parasite-infected red cell

71 is reflected in an altered asymmetry of lipid composition

72 in its cell surface membrane. In contrast to the uninfected,

73 healthy red cell, the malaria-infected red cell shows a

74 translocation of the anionic phosphatidylserine from the

75 inner leaflet to the outer leaflet of the bi-layer [13]. As a

76 result, the FITC-Annexin negative, healthy red cell now

77 turns to become FITC-Annexin positive [14]. Thus a

78 Plasmodium-infected red cell seems to mimic the anionic

79 surface charge that characterizes the bacterial cell surface.

80 In principle, this is expected to make malaria-infected red

81 cells become vulnerable to the action of anti-microbial82 peptides. Indeed, naturally occurring or modified peptides,

83 such as dermaseptin [15], oligoacyllysine [16], cyclosporin

84 A [17], cecropin A [18], NK-2 [14] and meucin [19], have

85 been found to displayin vitroanti-malarial activity. Some

86 membrane-active, hydrophobic peptides of fungal origin

87 have also been found to exhibit in vitro anti-malarial ac-

88 tion [20]. However, many of these naturally occurring pep-

89 tides suffer from drawbacks such as poor potency, stability

90 and selectivity [21]. Therefore, in a bid to improve their

91 performance, efforts are being made to engineer peptides

92 in diverse ways with the aim of reducing their size, im-

93 proving their stability against proteases and enhancing

94 their selectivity [22-24]. The structure activity relation-95 ships of a series of de novo-designed, conformationally-

96 constrained helical, amphipathic, cationic peptides against

97 bacteria have earlier been reported [25]. In the present

98 work, the potent anti-plasmodial action of these peptides

99 against both CQ-sensitive and CQ-resistant strains of P.

100 falciparumare being reported. The results indicate that a

101 lysine-branched, dimeric peptide Fd, which is highly po-

102 tent (IC501.39M) across CQ-sensitive and CQ-resistant

103 strains {Resistance Index (IC50 CQ resistant strain/ IC50104 CQ sensitive strain)1.1} of P. falciparum, fairly selective

105 against parasitized red blood cells {Selectivity Index (HC50

106URBC/IC50 P. falciparum) >35) and fairly non toxic to

107mammalian HeLa cells (TC50 >25 M), stalls parasite

108growth by causing arrest of ring stage parasite, anomalous

109egress of trophozoites and premature egress of schizonts

110that fail to produce invasion competent merozoites.

111Methods112Peptides

113Peptides Fm, (Fm)2, Fd, Fq, Fm, Fd, D-Lys- Fd,

114prochitinase and E30 (Table T11) were synthesized by

115Fmoc chemistry-based, manual, solid-phase synthesis.

116Didehydrophenylalanine (F) was chosen since it is a

117conformationally-constrained amino acid residue with a

118proven reputation to confer helical character to peptides.

119FITC derivatizations of (Fm)2and Fd were done after

120linking aminohexanoic acid to the N terminus. Peptides

121were purified to >95% homogeneity by RPHPLC and

122characterized by mass spectroscopy and circular dichro-123ism as described previously [25]. Chromatographic and

124mass spectral characterization is given as follows: RPHPLC

125profiles of control peptides prochitinase, E30 and bovine in-

126sulin (Additional file 1); Fm and Fd (Additional file 2)

127Electro Spray Mass Spectroscopy (ESMS) profiles of pro-

128chitinase, E30 and bovine insulin (Additional file 3); ESMS

129profiles ofFm and Fd (Additional file 4); MALDI of

130(Fm)2 (Additional file 5), RPHPLC and mass spectral

131data for Fq (Additional file6) and ESMS profiles of Fm,

132D-Lys- Fd and Fd (Additional file 7). Peptides corre-

133sponding to prochitinase, E30 (a 30 residues-long peptide

134from Hepatitis E virus ORF3) (synthesized and character- 135ized in house) and bovine insulin (Sigma) were used

136as controls in experiments on Fd mediated selective

137haemolysis of infected red cells. FITC-Insulin (Sigma)

138was used as control in experiments done to study the

139uptake of FITC tagged (Fm)2 and Fd by Plasmodium-

140infected RBCs.

141In vitrocultivation ofPlasmodium falciparum

142Chloroquine-sensitive (3D7) and CQ-resistant (Dd2 and

143INDO) strains ofP. falciparum were maintained in con-

144tinuous culture according to the method of Trager and

145Jensen [26] with minor modifications. Cultures were main-

146tained in fresh group O+ve human erythrocytes suspended147at 4% haematocrit in complete medium {16.2 g/L RPMI

1481640 containing 25 mM HEPES, 11.11 mM glucose

149(Gibco), 0.2% sodium bicarbonate (Sigma), 0.5% Albumax I

150(Gibco), 45 g/litre hypoxanthine (Sigma) and 50 g/litre

151gentamicin (Gibco)} and incubated at 37C under a gas

152mixture 5% O2, 5% CO2, and 90%N2. Every day, the spent

153medium was replaced by fresh complete medium to propa-

154gate the culture. For INDO strain in culture medium, albu-

155max was replaced by 10% pooled human serum (Innovative

156Research) as suggested by MR4 [27]. Parasitaemia was

157monitored by microscopic examination of Giemsa-stained

Kaushiket al. Malaria Journal2012,11:256 Page 2 of 15



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158 blood smears. Synchronized ring stage parasite was

159 obtained by 5% sorbitol treatment [28]. Trophozoites

160 and schizont-stage parasites were enriched by using

161 Percoll gradient [29].

162 Drug dilutions

163 Stock solutions of peptides and CQ were prepared in164 water (milli-Q grade) while artemisinin stock solution

165 was in dimethyl sulphoxide (DMSO). All stocks were

166 then diluted with culture medium to achieve the

167 required drug concentrations. The concentration of pep-

168 tide solution in water was based on A280 [E (M-1 cm-1)

169 F (didehydrophenylalanine) 19,000, W (Tryptophan)

170 5,000)]. Thus E280 were 62,000, 124,000, 124,000 and

171 248,000 for Fm, (Fm)2, Fd and Fq respectively.

172 The concentration of FITC-peptides was based on A495173 [E(M-1 cm-1) FITC 77,000 for (Fm)2with one FITC and

174 154,000 for Fd with two FITC per molecule]. Drugs

175and peptides solutions were placed in 96-well flat bot-

176tom tissue culture grade plates (Corning).

177Assay for anti-plasmodial activity

178For drug screening, SYBR green I based fluorescence

179assay was used as described previously by Smilkstein

180et al. [30]. Sorbitol synchronized ring stage parasites181(haematocrit: 2%, parasitaemia: 1%, 100 l) under nor-

182mal culture conditions were incubated in the absence or

183presence of increasing concentrations of peptides in

184water. CQ and artemisinin were used as positive con-

185trols. Vehicle control 0.4% DMSO (which was found to

186be non-toxic to parasite) was used in case of artemisinin.

187After 48 hr of incubation 100 l of SYBR Green I buffer

188[0.2 l of 10,000 X SYBR Green I (Invitrogen) per ml of

189lysis buffer {Tris (20 mM; pH 7.5), EDTA (5 mM),

190saponin (0.008%; wt/vol), and Triton X-100 (0.08%;

191vol/vol)}] was added to each well, mixed twice gently

t1:1 Table 1 In-vitro blood stage antiplasmodial activities, resistance and selectivity indices of peptides against different

t1:2 strains ofP. falciparum

t1:3 Peptides Peptide Sequence and design IC50P. falciparum(M) Resistance index HC50URBCMt1:4 3D7 Dd2 INDO IC50Dd2/ IC503D7

t1:

5 Fm Ac-GFRKFHKFWA-NH2

>100 >100 - >100t1:6 Fm Ac-GFRKFHKFWA-NH2 >100 >100 - - >100

t1:7Fd 1.39 0.1 1.6 0.09 1.5 0.075 1.15 >50 ( >35)*

t1:8D-Lys-Fd** 1.8 0.07 - - - >50 (> 27)

t1:9Fd 1.5 0.08 - - - >50 (>33)

t1:10 (Fm)2 Ac-GFRKFHKFWAAGFRKFHKFWA-NH2 2.4 0.15 2.5 0.13 - 1.04 >50 (>20)

t1:11

Fq 0.25 0.02 - - - 2.5 0.13 (10)

t1:12 Prochitinase EEPHKAASAEGKK > 40 - - - > 40

t1:13 E30 NPPDHSAPLGATRPSAPPLPHVVDLPQLGP > 40 - - - > 40

t1:14

InsulinGIVEQCCASVCSLYQLENYCN

FVNQHLCGSHLVEALYLVCGERGFFYTPKA

> 40 - - - > 40

t1:

15 Artemisinin 0.015 0.016 0.015 1

t1:16 Chloroquine 0.04 0.16 0.5 4

t1:17 * Hemolytic Selectivity index (HC50URBC/ IC50Pf3D7) is shown in parenthesis, **: KD refers to Lysine of D configuration. Values of standard deviation given as aret1:18 based on three independent observations.




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192 with multi-channel pipette and incubated in dark at 370C

193 for 1 h. Fluorescence was measured with a Victor fluores-

194 cence multi-well plate reader (Perkin Elmer) with excita-

195 tion and emission wavelength centred at 485 and 530 nm,

196 respectively. Fluorescence counts for CQ (0.1M for 3D7,

197 1 M for INDO) were subtracted from counts in each

198 well. The fluorescence counts were plotted against the

199 drug concentration and IC50 (the 50% inhibitory concen-

200 tration) was determined by analysis of doseresponse

201 curves. Results of the above mentioned fluorescence-

202 based assay were validated microscopically by examination

203 of Giemsa-stained smears of peptide-treated parasite cul-

204 tures. Statistical significance of relative potencies of pep-

205 tides was determined by students Ttest.

206 In vitro stage dependence of action

207 Stage specificity of action ofFd on the parasites blood

208 stage life cycle was determined by microscopic analysis of209 the effect ofFd on each of the three stages (ring, tropho-

210 zoite and schizont) of the parasite life cycle. Synchronized

211 stages were obtained by sorbitol-mediated synchronization

212 repeated thrice (synchronization 1, medium washed, incu-

213 bation for 3 hr, 370C, synchronization 2, medium washed

214 and culture allowed to grow in complete medium for

215 48 hr. At this stage the culture was synchronized a third

216 time to obtain highly synchronized ring stage culture).

217 This culture was grown for 24 hr and 38 hr to obtain

218 trophozoite and schizont stage cultures respectively. Both

219 trophozoite and schizont enriched cultures were subjected

220 to Percoll gradient centrifugation to obtain highly purified221 parasites of specific stages. Giemsa-stained smears were

222 microscopically observed over 2,000 RBCs to obtain dif-

223 ferential counts.

224 Cultures (1% parasitaemia, 2% haematocrit) at each of

225 the above mentioned stages were seeded in 96-well

226 plates containing different concentrations of Fd and

227 the plates incubated for 12 h (schizont), 24 h (trophozo-

228 ite) and 48 h (ring) under standard culture condition.

229 Smears were drawn, Giemsa-stained and analysed micro-

230 scopically. Stage-specificity of action was assessed by ob-

231 serving the stage transitions in drug-treated samples

232 against untreated controls.

233 Cytotoxic activity ofFd on HeLa cells using MTT assay

234 The cytotoxic effects of Fd on mammalian cells was

235 assessed by functional assay as described [31] using

236 HeLa cells cultured in RPMI containing 10% fetal bovine

237 serum, 0.21% sodium bicarbonate (Sigma) and 50 g/mL

238 gentamycin (complete medium). Briefly, cells (104 cells/

239 200 l/well) were seeded into 96- well flat-bottom tissue

240 culture plates in complete medium. Peptide solutions

241 were added after 24 hr of seeding and incubated for

242 48 hr in a humidified atmosphere at 37C and 5% CO2.

243 DMSO (as positive inhibitor) was added at 10%. Twenty

244microlitres of a stock solution of MTT (5 mg/mL in 1X

245phosphate buffered saline) was added to each well, gen-

246tly mixed and incubated for another 4 hr. After spinning

247the plate at 1500 rpm for 5 min, supernatant was

248removed and 100 l of DMSO (stop agent) was added.

249Formation of formazon was read on a microtiter plate

250reader (Versa max tunable multi-well plate reader) at

251570 nm. The 50% cytotoxic concentration (TC50) of drug

252was determined by analysis of doseresponse curves.

253Haemolysis assay

254Selectivity of haemolysis by peptides for infected ery-

255throcytes (PRBC) vs uninfected erythrocytes (URBC)

256was examined by incubating the test molecules with

257URBCs and PRBCs respectively in phosphate-buffered

258saline (PBS). Briefly, fresh RBCs were spin washed (1600

259RPM; 5 min) three times in PBS and re-suspended in

260PBS at 2% haematocrit. A 100 l suspension was added261to 96-well plate containing the peptides at different con-

262centrations. PBS alone (for baseline values) and 0.4%

263Triton X-100 in PBS (for 100% haemolysis) were used as

264controls. After incubation at 37C for 3 hr, the samples

265were centrifuged and supernatant was used to determine

266the haemolytic activity measured in terms of haemoglo-

267bin release as monitored by A415. Triton-treated control

268samples were diluted 10-fold before reading absorbance.

269Base line value (PBS control,


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296 and flooded with CY3 labelled anti-mouse antibody

297 (Sigma)(1: 500 dilution in 1% BSA/PBS,1 hr,370C in dark),

298 (d) PBS washed and flooded with DAPI (4, 6-diamidino-2-

299 phenylindole) (invitogen) (500 ng/ml, 10 min, 370C). After

300 a final PBS wash the smears were observed under Nikon

301 eclipse fluorescence microscope.

302 For studying peptide localization,P. falciparumcultures

303 were individually incubated with FITC-Fd (2M), FITC-

304 (Fm)2(2 M) or FITC-Insulin (3 M) a) alone and b) to-

305 gether with DAPI in complete medium at 37C for 30 min

306 and the cells were spin washed (1,600 RPM, 5 min) twice

307 with 1 X PBS to reduce background fluorescence. The

308 cells were smeared on a glass slide, and fluorescence was

309 visualized by using the respective filter settings for FITC

310 and DAPI.

311 For studying the selectivity and route of transport of

312 Fd into the red cell-resident malaria parasite, URBC and

313 PRBC were incubated with FITC-Fd (4 M) in parallel314 sets at 4C vs at room temperature (25C) for specified

315 times and spin washed (1,600 RPM, 2 min) with complete

316 medium (3 X 200 l). The cells were smeared on a glass

317 slide and both bright field images and fluorescence images

318 (using FITC filter) were captured at 100 X magnification

319 using Nikon eclipse fluorescence microscope. The soft-

320 ware Adobe Photoshop was used to overlay the fluores-

321 cence image on the bright field image.

322 Kinetics of peptide uptake

323 Kinetics of FITC-labelled peptide uptake was studied

324 using Flow cytometer (BD FACS callibur). FITC- Fd

325(3 M) was incubated for indicated time intervals with

326synchronized rings (~7% parasitaemia, 2% haematocrit)

327and synchronized trophozoites (~20% parasitaemia,

3282% haematocrit) stage cultures in a total volume of

329100 l. Cells were spin washed (1 min) with 1 ml

330PBS and samples injected into FACS.

331Results332Inhibition ofPlasmodium falciparum growth by peptides

333The anti-plasmodial activities of the de novo-designed,

334synthetic peptides Fm, Fm,Fd, D-Lys-Fd, Fd, (Fm)2335andFq (Table1), were determined by quantitative SYBR

336Green I based estimation of DNA replication after one

337developmental cycle (48 hr) as a measure of growth

338(see Figure F11 for growth inhibition profiles of Fm,

339(Fm)2, Fd and Fq). In contrast to the monomers

340Fm/Fm (IC50 >100 M), the dimers showed potent

341{IC50 : (Fm)2 2.4 M, Fd 1.39 M, D-Lys-Fd 1.8 M,342Fd 1.5 M} dose dependent anti-plasmodial action

343against the growth of CQ-sensitive, blood stage parasite

344(P. falciparum 3D7) in culture. Interestingly the K-K2345branched tetrameric dendrimer Fq with IC50 0.25 M

346turned out to be the most potent anti-plasmodial in

347the present series. The progressive increment in anti-

348plasmodial potency with valency of the peptides sug-

349gests an oligomeric state of the peptide is associated

350with potency. The haemolysis-based selectivity indices

351for the potent peptides were >35 (Fd), >20 (Fm)2 ,

352>27 (D-Lys-Fd), >33 (Fd) and 10 (Fq). The favourable

353index of >35 for Fd became the reason to study this

Peptide (M)

( )

( )

%Growth

100

(Fm (48 h)

(Fm)2(48 h)

(Fm)2(96 h)

Fd (48 h)

Fd (96 h)

Fq (48 h)

Figure 1Multivalent cationic, amphipathic helical peptides are potent inhibitors of the growth of malaria parasite in culture.

Dose-dependent effects ofFm (monomer), [(Fm)2 and Fd] (dimers) andFq (quadrumer) on the growth of ring-stage synchronized

Plasmodium falciparum(3D7) culture of malaria parasite. The anti-plasmodial potency increases in going from monomer Fm, to dimers [Fd,

and (Fm)2] and the quadrumer Fq. The marginal difference in the comparative growth inhibition profiles of the two dimers at 48 hr vs 96 hr

suggests that there is predominantly early death. Each data point represents the mean+/SD of three replicates.




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Ring Stage synchronized culture after 48 ha

Untreated Fd 1.56 M (IC50) 3.12 M (IC80)

Trophozoite stage synchronized culture after 24h

Untreated Fd 2.5 M (IC70)

Schizont stage Synchronized culture after 6 12 h.

0 h 9 h 12 h6h

C

B

A

Control

Fd2.5M

Figure 2Microscopy of anti-plasmodial action ofFd onPlasmodium falciparum 3D7. (A)Untreated or Fd-treated, ring-stage

synchronized cultures (parasitaemia 1%) were observed after 48 hr. Untreated culture shows high ring-stage parasitaemia, Fd IC50 and IC80treated cultures show low trophozoite-stage and low ring-stage arrested parasitaemia respectively, (B) Untreated orFd-treated trophozoite-stage

synchronized cultures were observed after 24 hr. Untreated culture shows intracellular rings while the Fd-treated culture shows anomalously

egressed trophozoites. Note the selectivity in action on parasitized cells with no effect on uninfected cells. (C)Untreated orFd-treated schizont stage

synchronized cultures were observed at 612 hr. While schizonts with the characteristic rosette arrangement of merozoites are intracellular at 6 hr in

untreated culture, they have (a) prematurely egressed and (b) lost the rosette arrangement of merozoites in the peptide treated culture (For zoom of

the images, see additional file10, panel A). At 12 hr while the merozoites in control have invaded fresh red cells to form rings, the merozoites of

peptide treated cultures have failed to invade and form rings (For quantitative account of decrease in invasion events , see additional file10, panel C).




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354 peptide in greater detail. Further, (Fm)2 was studied

355 since it was interesting to compare a linear dimer with a

356 branched dimer. The K-K2 dendrimeric quadrumer was

357 not studied in detail due to its poor haemolytic index. Sev-

358 eral control peptides (Table1) including sequences corre-

359 sponding to prochitinase, E30, and bovine insulin showed

360 no inhibition up to a concentration of 40M. Interestingly,

361 both (Fm)2and Fd retained their anti-plasmodial poten-

362 cies also against the CQ-resistant Dd2 strain resulting in re-

363 sistance index values of ~1 (Table 1). The more potent,

364 branched dimer Fd showed IC50value of 1.5 M against

365 the highly CQ-resistant INDO strain of P. falciparum.

366 These results showed that dimerization potentiates anti-

367 plasmodial activity by more than 50-fold over the corre-

368 sponding monomer. The small but significant differ-

369 ence (students T test p: 0.013) between the potencies

370 of the linear [IC50: (Fm)2 2.4 M] and branched

371 (IC50: Fd 1.39 M) dimers suggests that the mode372 of dimerization may also play a subtle role in modu-

373 lation of potency. When examined for comparative

374 potency in 48 hr (one cycle) vs 96 hr (two cycles)

375 assays, only marginal increments in potency [1.5 fold

376 (Fm)2, and 1.1 fold Fd] were observed at 96 hr

377 (Figure 1).

378 Ring vs trophozoite: selectivity in the action ofFd

379 In order to find whether there was ringvs trophozoite se-

380 lectivity in the action ofFd, microscopic evaluation of its

381 action was studied against parasitized red cells synchro-382 nized at ring (FigureF2 2A) and trophozoite (Figure 2B)

383 stages respectively. When the ring stage parasite culture

384 was treated with IC50 dose of Fd, it was observed

385 (Figure2A) that, after 48 hr of culture, the rings had pro-

386 gressed only up to the trophozoite stage suggesting bio-

387 chemical arrest and the resulting interception of the

388 progression to the schizont stage. Further at IC80, it was

389 observed that the rings did not mature even to the tropho-

390 zoite stage and the arrest of the parasite cycle was at

391 the ring stage. Since Fd at its IC50 caused arrest at

392 trophozoite stage (Figure 2A), the effect ofFd at its

393 IC70 (2.5 M) was tested on cultures synchronized at

394 trophozoite stage. It was interesting to see (Figure2B) that395 the peptide caused anomalous egress of trophozoites.

396 Even as 95% of trophozoites were found to be extra-

397 cellular (Additional file 8); this phenomenon was not

398 a consequence of non-specific haemolysis since uninfected

399 red cells were not affected (Figure 2B). Thus it appears

400 that at ~ IC80 rings are metabolically arrested and the

401 RBCs harbouring them are not lysed while such doses

402 cause selective lysis of parasitized RBCs that harbour tro-

403 phozoites. The observation of MSP3 staining in ~ 40% of

404 the anomalously egressed trophozoites (Additional file 9)

405 is worth noting.

406Fd is fairly non toxic to mammalian HeLa cells

407Toxicity of Fd to mammalian cells was examined by

408MTT assay. HeLa cells incubated with varying concen-

409trations (2.5-25 M) ofFd (Figure F33) did not show any

410toxicity. This suggests that the therapeutic index (TC50411Mammalian cells/IC50 P.falciparum) of this peptide

412(>16) is promising.

413Fd causes premature egress of undifferentiated Schizonts

414While it is unnatural for trophozoites to egress, the egress

415of schizonts is a natural process that leads to increased

416parasitemia. It was therefore interesting to find the effect

417ofFd on egress of schizonts. As shown (Figure2C), the

418peptide treated cultures showed premature egress at 6 h

419at a time when the schizonts in the control culture were

420intracellular. A close look at the schizonts of the control

421and the peptide treated cultures (see Additional file 10,

422panel A) revealed that (a) The characteristic symmetric423rosette arrangement of merozoites seen in control at 6 hr

424and 9 hr is absent in the prematurely egressed schizonts

425of the peptide treated culture and (b) the well differen-

426tiated merozoites of the control are invasion competent

427which enables them to form new rings while the mero-

428zoites of the peptide-treated culture are invasion disabled

429resulting in no new infections of red blood cells. Interest-

430ingly merozoites from both the control and peptide trea-

431ted schizonts were found to be MSP3+ (Additional file9).

432Selective haemolytic effect ofFd

433The anomalous egress of trophozoites via haemolysis 434motivated an examination of the selectivity in the action

435ofFd against parasitized(PRBC)vs uninfected red cells

436(URBC) over a range of peptide concentrations. As

437shown (Figure F44A), while the URBCs showed consider-

438able resistance to lysis, the PRBCs showed increasing

439lysis both with increasing concentration of peptide and

440also with increasing parasitaemia. It may be noted that

441the observed lysis is proportional to the percentage of

Figure 3Histogram showing results of MTT assay measuring

viability of HeLa cells incubated with Fd at different

concentrations. Data shows mean and standard deviation of three

independent observations.




9/16

442trophozoites in the cultures tested. Thus in the two

443mixed cultures shown in Figure 4A, the percentage

444haemolysis values of 7% and 16% correspond to percent-

445age trophozoite populations of ~ 7% and 15%, respect-

446ively. Further microscopic evaluation of mixed parasite

447cultures treated with Fd (12 M) revealed (Figure 4B)

448that only the trophozoites and not the rings were

449observed to be extracellular. In order to find if the

450observed lysis of infected cells was specific to Fd or

451would any peptide in general cause similar lysis,

452three control peptides (insulin, E30 and prochitinase)

453(Figure4A) were tested and found not to show any haem-

454olysis up to a concentration of 40 M. Microscopic exam-

455ination of ~ 2,000 cells from infected red cell cultures

456revealed a peptide concentration dependent inverse rela-

457tion between intracellular vs extracellular trophozoites

458(Figure4C). Also evident from this figure is the stability of

459ring-infected cells up to 12M ofFd.

460Anomalously egressed trophozoites are not surrounded

461by host cell membrane

462Immunostaining with band 3 antibody was done in order

463to find if the trophozoites egressed in response to Fd

464were free or packaged in host cell membrane. As shown

465in Figure F55, while the untreated culture showed the DAPI

A

B

C

Fd (M)

Trophs

(extracellular)

Rings(intracellular)

Trophs

(intracellular)

%Trophozoites/Rings

%Hemolysis

Fd

20% P

(15% Trophs)

Fd10% P

(7 % Trophs)

Fd

URBC

Insulin

10% P

Peptide (M)

Figure 4 Fd causes selective haemolysis of parasitized red

cells leading to anomalous egress of trophozoites. (A) Samples

of mixed stage parasite culture at different parasitaemia (P)

(% figures on respective curves) were incubated (3 hr) with the

indicated concentrations of peptide and percentage haemolysisestimated by A415. Control peptide (insulin) with infected red cells

(10% trophozoite-stage parasitaemia, solid line); Fd with URBC,

(dashed line); Fd with 10% parasitaemia (rings 3%, trophozoites 7%,

dashed dotted line); and Fd with 20% parasitaemia (rings 5%,

trophozoites 15%, dotted line). Two other control peptides

(prochitinase and E30) behaved like insulin (data not shown).

(B)Shows microscopic analysis of the selective sensitivity of

trophozoites (, anomalously egressed)vs rings (*, intracellular) at

12MFd,(C) shows dose-dependent selective effect ofFd on

anomalous egress of trophozoites but not rings, monitored

microscopically after incubation (3 hr). Data shown were obtained

after counting 2,000 erythrocytes.

Figure 5Fd-mediated parasite egressed from red blood cells

are not coated with host cell membrane. Bright field optical

images (top panel) show haemozoin crystals that are intracellular in

control and appear to be extracellular inFd (12.5M)-treated

sample. Panel 2 shows DAPI stained nuclei of the malaria parasite.

Panel 3 (immunostaining with band 3 antibody) indicates that band

3 (red) was seen in all cells. Panel 4 (overlay of DAPI and band 3)

indicates that the parasites (staining blue) in control panel are

intracellular and flanked by band 3 stain. But the Fd-treated

parasites, which egressed anomalously, are extracellular and not

flanked by band 3.




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466 stained parasites to be intracellular and flanked by band 3

467 staining (red), the egressed extracellular trophozoites in

468 the Fd-treated culture (12.5 M) had no band 3 staining

469around them. This observation suggests that egress does

470not involve host cell membrane and is likely to be

471mediated via lysis of the host cell.

A

B

C

Figure 6Cellular localization of FITC-labelled peptides in Plasmodium falciparum-infected red blood cells. (A) FITC-Fd (3 M) was

incubated with parasitized culture (30 min, 37C). Fluorescence image overlaid on optical image shows that FITC-Fd exhibits selective entry into

parasitized RBCs. Arrow heads:red (trophozoites showing haemozoin), blue (likely to be ring stages with low fluorescence). ( B) FITC-Fd and FITC-

(Fm)2 but not FITC-insulin are internalized by PRBC. The overlay of FITC fluorescence (green) with DAPI (blue) suggests that these two peptides

bind to DNA of the parasite. (C)Transport of FITC-Fd (4 M) from RBC surface to parasite: Fd has selective affinity for infected RBC (PRBC)

surface. The slow entry of FITC-Fd into PRBC at 4C becomes fast at 25C.




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472 Dimers Fd and (Fm)2show selective penetration into

473 Plasmodium-infected RBCs

474 To gain a better understanding of the anti-plasmodial

475 action of the two dimeric peptides, localization of

476 peptides was studied using fluorophore-labelled pep-

477 tides. Fluorescence microscopy with FITC-labelled Fd

478 showed that this peptide was selective in targeting the

479 parasite inside the infected RBC (Figure 6A). Co-

480 localization of FITC florescence (green) with DAPI flor-

481 escence (blue) (Figure 6B) indicated that the two pep-

482 tides bind to the DNA of the malaria parasite. In order

483 to check whether entry of the two dimers was specific or

484 would any other peptide also enter parasitized cells,

485 FITC-labelled insulin was examined for uptake by the

486 parasitized cells. Fluorescence microscopy revealed that

487 there was no accumulation of FITC-insulin in parasitized

488 cells suggesting specificity in uptake and anti-plasmodial

489 action of the two dimeric peptides. Since no fluorescence490 was observed on the red cell surface even as there was

491 intense fluorescence intracellularly, it was surmised that

492 the uptake of the peptide may be faster than the time

493 (30 min) given for the experiment. In order to capture

494 early events in transfer of the peptide from the RBC sur-

495 face to the parasite, a comparative uptake study at 4C

496 vs at 25C was performed. As shown (Figure 6C), while

497 the URBC showed no staining, the PRBC at 4C showed

498 predominantly surface staining with a modicum of intra-

499 cellular staining. However PRBC at 25C showed a tran-

500 sition from surface to intracellular staining at 10 min

501 which became completely intracellular at 30 min. Thus

502it appears that parasite-infected red blood cells are well

503geared for rapid uptake of this peptide.

504Uptake kinetics ofFd

505In order to assess the kinetics of uptake of the fluores-

506cently tagged peptide into the infected red cells, a time-

507dependent analysis of the phenomenon was studied by

508FACS. Monitoring the uptake in ring-synchronized cul-

509tures (Figure F77) revealed a low uptake (1.97%) at the first

510minute rising to ~3% at 20 min. In contrast to rings, the

511analogous peptide uptake by trophozoites was found to

512be fast at the very first minute (7.6%) with further sub-

513stantial rise to 22% at 20 min.

514Discussion515The success of antibiotics is based upon the characteris-

516tic molecular targets that distinguish the prokaryotic

517bacteria from the nucleated eukaryotic cells [32,33]. Cat-518ionic, amphipathic helical, antibiotic peptides also seem

519to gain specificity by exploiting the fact that bacteria

520have a preponderance of anionic lipids, such as phospha-

521tidylglycerol and bis(phosphatidyl)glycerol (cardiolipin),

522conferring a negative charge on their surface. In con-

523trast, their eukaryotic counterparts have a high density

524of zwitterionic lipids such as phosphatidylcholine and

525phosphatidylethanolamine, enabling their surfaces to be

526largely neutral [34,35]. A well-studied and yet curious

527feature of the human red blood cell is the transition

528from FITC-Annexin negative to FITC-Annexin positive

529status upon infection with the malaria parasite [14]. It is

Figure 7Kinetics ofFd entry into parasitized red blood cells (synchronized rings (~7% parasitaemia) and synchronized trophozoites

(~20% parasitaemia).Uptake of fluorescently tagged FITC-Fd (2.5 M) was monitored by flow cytometry at 25C. Panels A and B depict the

peptide uptake profiles obtained with rings and trophozoites, respectively. Zero minute profiles correspond to samples not treated with peptide.

Figures against percentage gated indicate the number of cells stained above the threshold line. Note (a) the fast uptake and progressive increase

in the number of fluorescent signals with time, and (b) faster uptake by trophozoites compared to ring-stage parasitized cells.




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530 well known that this phenomenon is caused by the

531 translocation of the anionic phosphatidylserine from the

532 inner to the outer leaflet of the lipid bilayer. Thus infec-

533 tion with Plasmodium confers an anionic character to

534 the red blood cell giving it a shade of semblance to a

535 bacterial membrane. Focusing on the altered membrane

536 asymmetry seen in the infected red cell, the interesting

537 anti-plasmodial properties of several de novo-designed,

538 cationic, amphipathic, helical, bonafide membrane-active

539 anti-bacterial peptides have been examined in the

540 present studies.

541 The first observation of the comparative anti-plasmodial

542 potencies of two monomeric (Fm, Fm) and four di-

543 meric peptides {Fd, Fd, D-Lys-Fd, (Fm)2}(Table 1)

544 indicated that the dimers (IC50 1.39- 2.4 M) were

545 about two orders of magnitude more potent than the

546 monomers (IC50 >100 M). Among the dimeric pep-

547 tides {Fd: IC50 1.39 M, D-Lys-Fd: IC50 1.8 M,548 (Fm)2: IC50 2.4 M, Fd: IC50 1.5 M}, the lysine-

549 branchedFd was chosen for detailed mechanistic stud-

550 ies since it had favourable features of anti-plasmodial

551 potency and selectivity index (>35). Interestingly, in

552 going from this bivalent-branched dimerFd to the tetra-

553 valent K-K2-branched quadrumer Fq (IC50: 0.25 M), a

554 further six-fold potentiation was observed. However, as

555 shown in Table1, this potentiation was associated with a

556 decline in selectivity index from >35 (Fd) to 10 (Fq).

557 Nevertheless, the trend of increasing anti-plasmodial po-

558 tency with increasing valency (monomer to dimer to

559 quadrumer) of the peptide suggests that oligomerization560 on cell surfaces may play an important role in the anti-

561 plasmodial action of these cationic, amphipathic peptides.

562 It is important to note that crystal structures of several

563 F-containing peptides have revealed the propensity of

564 this planar aromatic residue to engage in long-range, mul-

565 ticentred interactions (N-H. . .O, C-H. . .O, C-H. . ., and

566 N-H. . .) that can stabilize oligomeric states like the F

567 zipper [36] in the absence of linker, or the helical hairpins

568 in the presence of appropriate linker [37,38]. The coming

569 together of optimal values of anti-plasmodial potency and

570 selectivity indices (haemolytic selectivity index >35 and

571 mammalian cell cytotoxicity index >16) in the lysine-

572 branched dimeric Fd became the motivation to unravel573 mechanistic details of the anti-plasmodial action of this

574 peptide. (Fm)2,the corresponding linear dimer, was also

575 studied in some experiments to explore if the mode of

576 dimerization may influence the anti-plasmodial actions of

577 these two dimeric peptides.

578 The essentiality of apicoplast, an organelle of cyano-

579 bacterial origin in the malaria parasite, is well known to

580 make the parasite vulnerable to antibiotics,such as tetra-

581 cycline, clindamycin and thiostrepton [39,40], which are

582 known to cause delayed death in malaria parasite. This

583 phenomenon, caused by targeting of the apicoplast or

584the mitochondrion, is characterized by a significantly

585lower IC50 post second cycle (at 96 hr) vs the first cycle

586(at 48 hr) [41]. In order to find if the anti-plasmodial

587peptides under study may be targeting organelles such

588as the apicoplast of the malaria parasite, comparative

589anti-plasmodial potencies against P. falciparum 3D7

590were determined at both 48 hr and 96 hr. Since the data

591(Figure1) did not show a significant reduction of IC50 at

59296 hr, the possibility that Fd and (Fm)2may cause early

593death by influencing several other targets besides the api-

594coplast and the mitochondria cannot be ruled out.

595In trying to gain a better understanding of the prob-

596able mechanisms that confer the malaria parasite growth

597inhibitory properties on the dimeric peptide Fd, the

598peptide-treated samples were examined by microscopy.

599As shown (Figure2A), in comparison to the untreated con-

600trol (high ring-stage parasitaemia), while the IC50-treated

601ring stage synchronized culture was found to have the ini-602tial low parasitaemia (1%) and growth arrest at trophozoite

603stage, the IC80treated sample was found to have the intial

604parasitaemia (1%) with the few parasitized cells showing

605arrested, probably dead pyknotic ring forms. Interest-

606ingly, the microscopic examination of the Fd-treated,

607trophozoite-enriched culture (Figure2B) showed the pres-

608ence of extra erythrocytic Giemsa-positive trophozoites

609alongside uninfected red blood cells. Indeed manual

610counting of a large number of fields (Additional file 8)

611indicated that over 95% of the trophozoites were in fact

612extracellular. The presence of extracellular trophozoites in

613the midst of intact uninfected red blood cells was suggest- 614ive of the selective haemolytic action ofFd on parasitized

615cells causing anomalous release of trophozoites following

61624-hr incubation.

617The transition of ring stage to trophozoite stage in

618presence ofFd at its IC50, indicates that while this low

619dose is sufficient to arrest trophozoites, it is clearly not

620sufficient to halt the ring from moving to the trophozo-

621ite stage (Figure 2A). This heightened sensitivity of

622trophozoite-stage cultures over ring-stage cultures may

623be related to the enormous red cell reorganizational

624changes associated with a fast feeding, actively metabol-

625izing and replicating life style of trophozoite in compari-

626son with the more sedentary ring stage. The selective627lysis of trophozoite-bearing cells (Figure 4B) also sug-

628gests that such remodelling of the trophozoite harbour-

629ing red cell membrane [42] may be rendering it more

630vulnerable to the action of membrane active peptides

631like the Fd. The greater vulnerability of trophozoite

632bearing over ring-bearing red cells is evident also from

633the fact that peptide-mediated haemolysis is directly pro-

634portional to the percentage trophozoites in mixed stage

635culture samples (Figure4A).

636Trophozoite egress, induced by the peptide, is not nat-

637ural to the life cycle of the malaria parasite. Hence, it




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638 was important to find if host cell membranes-may be

639 involved in the process. To address this issue, the

640 peptide-treated sample was exposed to immunostaining

641 with band 3 antibody. As shown (Figure 5), the egressed

642 trophozoites were not flanked by band 3 staining sug-

643 gesting that the process is more likely to be caused by

644 lysis of the infected host cell. A closer examination of

645 the phenomenon ofFd-mediated anomalous egress of

646 trophozoites revealed that in trophozoite-ring mixed cul-

647 ture exposed to FITC-Fd at low concentrations (2 M)

648 and short time (30 min) (Figure 5A), the peptide seems

649 to enter and attack the parasite from within without

650 causing immediate lysis of the infected red cell. However

651 when trophozoite-stage culture was exposed to Fd for

652 longer times (24 hr), at similar low concentrations

653 (2 M), this peptide seemed to cause selective lysis of

654 parasitized cells leading to anomalous egress of tropho-

655 zoites (Figure 2B). Further, at higher concentrations656 (12.0 M) this peptide caused selective lysis of red cells

657 harbouring trophozoites within 3 hr (Figure4B).

658 Unlike trophozoites, schizonts have an intrinsic pro-

659 gram of egress that causes the release of numerous mer-

660 ozoites leading to infection of fresh red cells causing

661 amplification of infection and increasing the severity of

662 disease. Hence it was interesting to find ifFd may per-

663 turb the programmed process of egress in schizonts. As

664 shown (Figure 2C and Additional file 10), this peptide

665 caused premature egress of schizonts. As a consequence,

666 the egressed schizonts which showed lumps of amplified

667 DNA did not exhibit the characteristic symmetrically668 organized rosette appearance of merozoites seen in the

669 untreated control schizont. Further the merozoites from

670 the peptide treated culture showed a significantly low in-

671 vasion efficiency in comparison to the control mero-

672 zoites (Additional file 10). FigureF8 8 summarizes the

673 versatility ofFd to target each stage of the life cycle of

674 P. falciparum in characteristic and decisive ways with

675 good selectivity.

676 Malaria parasites go to extraordinary means to modify

677 RBC membrane, which separates them from the external

678 world. These modifications include a marked increase in

679 erythrocyte membrane fluidity [43-46], alterations in

680 host cell lipid fatty acid composition [47,48] and681 phospholipid-transbilayer distribution [49], enhancement

682 of the rate of lipid transbilayer movement [50,51] and

683 increased permeability through newly formed pores on

684 the erythrocyte membranes [52,53]. As a part of these

685 major re-organizational events, the malaria-infected red

686 cell is well known to exhibit a translocation of the an-

687 ionic phosphatidylserine from the inner leaflet to the

688 outer leaflet of the bi-layer [13,54]. This more negative

689 cell surface may provide the force for the fast and spe-

690 cific uptake of cationic peptides by the malaria-infected

691 red cell. Previous studies have indicated that high levels

692of cellular uptake can be achieved through the inclusion

693of cationic residues into arginine-based peptide oligo-

694mers [55]. The positive molecular charge facilitates

695charge-driven uptake through the plasma membrane,

696which exhibits a potential gradient that can electrophor-

697ese cationic species from the extracellular space into the

698cell [56,57]. Interestingly, a recent study has demon-

699strated that membrane asymmetry can be altered and

700maintained in the altered state by externally added poly-

701L-lysine [58]. The combined microscopic (Figure 6) and702FACS analysis (Figure 7) suggests that Fd enters the

703infected cells and stains rings and trophozoites within a

704few minutes. Thus it is quite likely that Fd and (Fm)2,

705the two cationic dimeric peptides studied here, in close

706resemblance to poly-L-lysine, may first home on those

707infected red blood cells that show slightly more anionic

708character as a result of alterations in membrane asym-

709metry and binding of these cationic peptides could

710further enhance and maintain this anionic character

711facilitating the stronger binding and faster internalization

712of peptides into the infected cells.

Figure 8Model of antiplasmodial action ofFd.Fd causes

growth arrest of rings, anomalous egress of trophozoites and

premature egress of schizonts. Its 1 C80 (3.12M) and IC50(1.56M) cause arrest of rings and trophozoites respectively and its

IC70 (2.5M) causes the anomalous egress of trophozoites and

premature egress of schizonts. In both cases the parasite fails to

proliferate since egressed trophozoites cannot differentiate into

schizonts and the premature, undifferentiated egressed schizonts

seem to release merozoites that are invasion incompetent. Thepeptide shows good selectivity against parasitized RBCs since 16X

IC80 fails to lyse healthy RBCs.




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713 The ability of Fd to cross the host red cell mem-

714 brane, the parasitophorous vacuole membrane, the para-

715 site plasma membrane and also the parasite nuclear

716 membrane to reach the nucleus of the parasite

717 (Figure6B), indicates its resemblance to cell-penetrating

718 peptides which are known to have a lipophilic-cationic

719 character. Even as the peptide was apparently targeting

720 the DNA of the parasite, the absence of FITC-Fd on

721 the host red cell membrane or all the subsequent mem-

722 branes mentioned above was puzzling. It was surmised

723 that these localizations may have been missed due to the

724 rapidity of the process of peptide uptake. In order to

725 capture some stages preceding the intranuclear entry of

726 the peptide, the peptide-staining experiment was per-

727 formed as a function of both time and temperature. As

728 shown (Figure6C), the images captured at 4C (30 min)

729 indeed showed predominant staining on the host cell

730 surface. In contrast, the images corresponding to 25C731 (10 min) and 25C (30 min) showed progressively

732 greater staining of the intracellular parasite nuclear ma-

733 terial. These results suggest that this peptide crosses

734 several membranes of the infected red cells before

735 entering the nucleus.

736 The most probable reasons for the significantly

737 enhanced potency of the dimersFd/Fd over the mono-

738 mers Fm/Fm include increased membrane binding

739 and permeabilization, enhanced binding affinity for

740 DNA and proteins and enhanced biochemical stability

741 against degradation by proteases. These properties ori-

742 ginating from increased avidity and affinity of inter-743 actions unique to dimeric peptides and absent in

744 monomeric peptides have been described previously

745 [25]. In studies on the antibiotic action of these peptides it

746 has previously been observed that the requirements of heli-

747 city for potent antibiotic action are much higher for the

748 gram positive Staphylococcus.aureus than is the case with

749 the Gram-negative Escherichia coli. In contrast, as shown

750 in the present study, all dimers {(Fd, Fd, D-Lys-Fd,

751 (Fm)2} are nearly equipotent against P. falciparum

752 (Table 1). This suggests that different conformational and

753 topological properties of peptides may be important for

754 their activity against different organisms.

755 Some important features of these peptides as drugs756 against malaria include their favourable resistance indices

757 (Table1) that allow them to rapidly kill both drug-sensitive

758 and drug-resistant strains of malaria parasite with equal po-

759 tencies, their amphipathic nature that gives them drug-like

760 character, and their ability to permeabilize and penetrate

761 biological membranes, which allows them to attack target

762 cells both from the surface as well as intracellularly. In

763 addition, the presence of the conformationally constrained,

764 non-protein, amino acid didehydrophenylalanine in both

765 Fd and (Fm)2 provides considerable protection against

766 proteolytic degradation [25]. Even as these two dimeric

767peptides offer similar profiles of anti-plasmodial actions, a

768judicious choice for further improvisation should be the

769branched dimer Fd over the linear dimer (Fm)2 since

770(a) the former is little more potent against P. falciparum,

771(b) the branched dimer is more stable against proteases

772[25], and (c) the branched dimer has better economics of

773production since the time it takes to synthesize a

774branched dimer is half as much as the time it takes to

775assemble a linear dimer.

776Conclusion777This study reports the anti-plasmodial action ofFd, a

778de novo-designed, cationic, lysine-branched amphipathic,

779helical peptide. In vitro assays suggest good selectivity

780(>35), good resistance index (1.1) and low mamamalian

781cell cytotoxicity, as a promise of Fd against malaria.

782The strategy adopted by Fd to inhibit the growth of783malaria parasite appears to be broadly two-fold: (a) in-

784volving growth arrest without causing lysis of red cell

785(at IC50-IC100), and (b) anomalous egress of tropho-

786zoites and premature egress of undifferentiated schi-

787zonts leading to death of the parasite (at> IC100).

788Additional files789

791Additional file 1: RPHPLC profiles of control peptides.

792Additional file 2: RPHPLC profiles ofFm and Fd.

793Additional file 3: ESMS of RPHPLC purified prochitinase, E30794and Insulin.

795Additional file 4: ESMS of RPHPLC purified Fm and Fd.796Additional file 5: MALDI mass spectrum (Bruker Daltonics Flex797analysis) of RPHPLC purified linear dimeric peptide.

798Additional file 6: Chromatographic and mass spectral799characterization ofFq.

800Additional file 7: ESMS of RPHPLC purified Fm, Fd and D-Lys-Fd.

801Additional file 8: Microscopic differential counts ofFd (2.5 M)802treated trophozoites after 24 h.

803Additional file 9: Fd treated schizonts express MSP3.

804Additional file 10: Fd causes premature egress of schizonts.

805Abbreviations806CQ: Chloroquine;F: Didehydrophenylalanine;Fm:F containing807monomeric decapeptide; Fd: Lysine branched dimer ofFm; (Fm)2: Linear

808dimer ofFm; DAPI: 4',6-diamidino-2-phenylindole; FACS: Fluorescence809activated cell sorter; FITC: Fluorescein isothiocyanate;810P. falciparum:Plasmodium falciparum; IC100: Inhibitory concentration causing811100% inhibition of growth; PRBC: Parasitized red blood cell; URBC: Uninfected812red blood cell; Troph: Trophozoite;Fq:813The K-K2dendrimer presenting a quadrumer form ofFm.

814Competing interests815The authors declare that they have no competing interests.

816Authorscontributions817NKK and JS carried out the experiments to determine the antiplasmodial818potencies of different peptides, NKK performed mechanistic experiments819including FACS and immunofluorescence microscopy, DS conceived of the820study, participated in its design, coordination and brain storming and drafted821the manuscript. All authors read and approved the final manuscript.


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822 Acknowledgements823 We thank MR4 who generously provided the chloroquine-resistant Dd2 and824 INDO strains used in the study. Thanks to X Su and the late Dr. David825 Walliker who deposited these strains with MR4, BEI Resources Repository,826 NIAID, NIH:. Our thanks to the anonymous reviewers for their critical and827 thoughtful comments that have enriched the manuscript enormously. We

828 thank Dr. Pawan Malahotra for anti-band 3 antibody, Sumit Rathore for FACS829 analysis, Dr. Maryam Imam for providing anti MSP3 antibody, Dr. Aparna830 Anantharaman for MTT assay and Dr.Anil Sharma for help with statistical831 analysis. NKK thanks Indian Council for Medical Research (ICMR), New Delhi,832 for Senior Research fellowship. We thank the ICGEB, New Delhi for internal833 funding.

834 Received: 8 May 2012 Accepted: 13 July 2012835 Published: 1 August 2012

836 References1.837 Tinto H, Rwagacondo C, Karema C, Mupfasoni D, Vandoren W, Rusanganwa

838 E, Erhart A, Van Overmeir C, Van Marck E, D'Alessandro U:In-vitro839 susceptibility ofPlasmodium falciparum to monodesethylamodiaquine,840 dihydroartemisinin and quinine in an area of high chloroquine841 resistance in Rwanda.Trans R Soc Trop Med Hyg 2006,100:509514.

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Anti-plasmodial Action of de Novo-Designed, Cationic, Lyisne Branched Amphipathic Helical Peptides