Upload
dineshmkm
View
218
Download
0
Embed Size (px)
Citation preview
7/27/2019 Anti-plasmodial Action of de Novo-Designed, Cationic, Lyisne Branched Amphipathic Helical Peptides
1/16
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
7/27/2019 Anti-plasmodial Action of de Novo-Designed, Cationic, Lyisne Branched Amphipathic Helical Peptides
2/16
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
http://www.malariajournal.com/content/11/1/256
mailto:[email protected]://creativecommons.org/licenses/by/2.0http://creativecommons.org/licenses/by/2.0mailto:[email protected]7/27/2019 Anti-plasmodial Action of de Novo-Designed, Cationic, Lyisne Branched Amphipathic Helical Peptides
3/16
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
http://www.malariajournal.com/content/11/1/256
7/27/2019 Anti-plasmodial Action of de Novo-Designed, Cationic, Lyisne Branched Amphipathic Helical Peptides
4/16
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.
Kaushiket al. Malaria Journal2012,11:256 Page 3 of 15
http://www.malariajournal.com/content/11/1/256
7/27/2019 Anti-plasmodial Action of de Novo-Designed, Cationic, Lyisne Branched Amphipathic Helical Peptides
5/16
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,
7/27/2019 Anti-plasmodial Action of de Novo-Designed, Cationic, Lyisne Branched Amphipathic Helical Peptides
6/16
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.
Kaushiket al. Malaria Journal2012,11:256 Page 5 of 15
http://www.malariajournal.com/content/11/1/256
7/27/2019 Anti-plasmodial Action of de Novo-Designed, Cationic, Lyisne Branched Amphipathic Helical Peptides
7/16
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).
Kaushiket al. Malaria Journal2012,11:256 Page 6 of 15
http://www.malariajournal.com/content/11/1/256
7/27/2019 Anti-plasmodial Action of de Novo-Designed, Cationic, Lyisne Branched Amphipathic Helical Peptides
8/16
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.
Kaushiket al. Malaria Journal2012,11:256 Page 7 of 15
http://www.malariajournal.com/content/11/1/256
7/27/2019 Anti-plasmodial Action of de Novo-Designed, Cationic, Lyisne Branched Amphipathic Helical Peptides
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.
Kaushiket al. Malaria Journal2012,11:256 Page 8 of 15
http://www.malariajournal.com/content/11/1/256
7/27/2019 Anti-plasmodial Action of de Novo-Designed, Cationic, Lyisne Branched Amphipathic Helical Peptides
10/16
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.
Kaushiket al. Malaria Journal2012,11:256 Page 9 of 15
http://www.malariajournal.com/content/11/1/256
7/27/2019 Anti-plasmodial Action of de Novo-Designed, Cationic, Lyisne Branched Amphipathic Helical Peptides
11/16
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.
Kaushiket al. Malaria Journal2012,11:256 Page 10 of 15
http://www.malariajournal.com/content/11/1/256
7/27/2019 Anti-plasmodial Action of de Novo-Designed, Cationic, Lyisne Branched Amphipathic Helical Peptides
12/16
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
Kaushiket al. Malaria Journal2012,11:256 Page 11 of 15
http://www.malariajournal.com/content/11/1/256
7/27/2019 Anti-plasmodial Action of de Novo-Designed, Cationic, Lyisne Branched Amphipathic Helical Peptides
13/16
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.
Kaushiket al. Malaria Journal2012,11:256 Page 12 of 15
http://www.malariajournal.com/content/11/1/256
7/27/2019 Anti-plasmodial Action of de Novo-Designed, Cationic, Lyisne Branched Amphipathic Helical Peptides
14/16
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.
Kaushiket al. Malaria Journal2012,11:256 Page 13 of 15
http://www.malariajournal.com/content/11/1/256
http://www.biomedcentral.com/content/supplementary/1475-2875-11-256-S1.pdfhttp://www.biomedcentral.com/content/supplementary/1475-2875-11-256-S2.pdfhttp://www.biomedcentral.com/content/supplementary/1475-2875-11-256-S3.pdfhttp://www.biomedcentral.com/content/supplementary/1475-2875-11-256-S4.pdfhttp://www.biomedcentral.com/content/supplementary/1475-2875-11-256-S5.pdfhttp://www.biomedcentral.com/content/supplementary/1475-2875-11-256-S6.pdfhttp://www.biomedcentral.com/content/supplementary/1475-2875-11-256-S7.pdfhttp://www.biomedcentral.com/content/supplementary/1475-2875-11-256-S8.pdfhttp://www.biomedcentral.com/content/supplementary/1475-2875-11-256-S9.pdfhttp://www.biomedcentral.com/content/supplementary/1475-2875-11-256-S10.pdfhttp://www.biomedcentral.com/content/supplementary/1475-2875-11-256-S10.pdfhttp://www.biomedcentral.com/content/supplementary/1475-2875-11-256-S9.pdfhttp://www.biomedcentral.com/content/supplementary/1475-2875-11-256-S8.pdfhttp://www.biomedcentral.com/content/supplementary/1475-2875-11-256-S7.pdfhttp://www.biomedcentral.com/content/supplementary/1475-2875-11-256-S6.pdfhttp://www.biomedcentral.com/content/supplementary/1475-2875-11-256-S5.pdfhttp://www.biomedcentral.com/content/supplementary/1475-2875-11-256-S4.pdfhttp://www.biomedcentral.com/content/supplementary/1475-2875-11-256-S3.pdfhttp://www.biomedcentral.com/content/supplementary/1475-2875-11-256-S2.pdfhttp://www.biomedcentral.com/content/supplementary/1475-2875-11-256-S1.pdf7/27/2019 Anti-plasmodial Action of de Novo-Designed, Cationic, Lyisne Branched Amphipathic Helical Peptides
15/16
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.
2.842 Mu J, Ferdig MT, Feng X, Joy DA, Duan J, Furuya T, Subramanian G, Aravind843 L, Cooper RA, Wootton JC, Xiong M, Su XZ:Multiple transporters844 associated with malaria parasite responses to chloroquine and quinine.845 Mol Microbiol2003,49:977989.
3.846 Jacqueline R:Halt called on single-drug antimalarial prescriptions. Nature847 2006, doi:10.1038/news060116-13.
4.848 Dondorp AM, Nosten F, Yi P, Das D, Phyo AP, Tarning J, Lwin KM, Ariey F,849 Hanpithakpong W, Lee SJ, Ringwald P, Silamut K, Imwong M, Chotivanich K,850 Lim P, Herdman T, An SS, Yeung S, Singhasivanon P, Day NP, Lindegardh N,851 Socheat D, White NJ:Artemisinin resistance in Plasmodium falciparum852 malaria. N Engl J Med2009,361:455467.
5.853 White NJ:Artemisinin resistance--the clock is ticking. Lancet2010,854 376:20512052.
6.855 Crompton PD, Pierce SK, Miller LH:Advances and challenges in malaria856 vaccine development.J Clin Invest2010,120:41684178.
7.857 WHO:World Malaria Report 2011, Issue Dec. 2011.http://www.who.int/858 mediacentre/factsheets/fs094/en/index.html.
8.859 Yount NY, Yeaman MR:Emerging themes and therapeutic prospects for860 anti-infective peptides.Annu Rev Pharmacol Toxicol2012,52:337360.
9.861 Yeaman MR, Yount NY:Mechanisms of antimicrobial peptide action and862 resistance. Pharmacol Rev2003,55:2755.
10.863 Epand RM, Shai Y, Segrest JP, Anantharamaiah GM:Mechanisms for the864 modulation of membrane bilayer properties by amphipathic helical865 peptides.Biopolymers1995,37:319338.
11.866 Feder R, Dagan A, Mor A: Structure-activity relationship study of867 antimicrobial dermaseptin S4 showing the consequences of868 peptide oligomerization on selective cytotoxicity. J Biol Chem 2000,869 275:42304238.
12.870 Zasloff M:Antimicrobial peptides of multicellular organisms. Nature2002,871 415:389395.
13.872 Sherman IW, Prudhomme J, Tait JF:Altered membrane phospholipid873 asymmetry inPlasmodium falciparum-infected erythrocytes.874 Parasitol Today1997,13:242243.
14.875 Gelhaus C, Jacobs T, Andra J, Leippe M:The antimicrobial peptide NK-2,876 the core region of mammalian NK-lysin, kills intraerythrocytic877 Plasmodium falciparum.Antimicrob Agents Chemother2008,52:17131720.
15.878 Ghosh JK, Shaool D, Guillaud P, Ciceron L, Mazier D, Kustanovich I, Shai Y,879 Mor A:Selective cytotoxicity of dermaseptin S3 toward intraerythrocytic880 Plasmodium falciparumand the underlying molecular basis. J Biol Chem881 1997,272:3160931616.
16.882 Radzishevsky I, Krugliak M, Ginsburg H, Mor A:Antiplasmodial activity of883 lauryl-lysine oligomers.Antimicrob Agents Chemother2007,51:17531759.
17.884 Azouzi S, El Kirat K, Morandat S:The potent antimalarial drug cyclosporin885 A preferentially destabilizes sphingomyelin-rich membranes.Langmuir886 2010,26:19601965.
18.887 Boman HG, Wade D, Boman IA, Wahlin B, Merrifield RB:Antibacterial and888 antimalarial properties of peptides that are cecropin-melittin hybrids.889 FEBS Lett1989,259:103106.
19. 890Gao B, Xu J: Rodriguez Mdel C, Lanz-Mendoza H, Hernandez-Rivas R, Du891W, Zhu S: Characterization of two linear cationic antimalarial peptides in892the scorpion Mesobuthus eupeus. Biochimie2010,92:350359.
20. 893Nagaraj G, Uma MV, Shivayogi MS, Balaram H:Antimalarial activities of894peptide antibiotics isolated from fungi. Antimicrob Agents Chemother2001,89545:145149.
21. 896Hancock RE, Sahl HG:Antimicrobial and host-defense peptides as new897anti-infective therapeutic strategies. Nat Biotechnol2006,24:15511557.
22. 898Dathe M, Wieprecht T: Structural features of helical antimicrobial899peptides: their potential to modulate activity on model membranes and900biological cells. Biochim Biophys Acta1999,1462:7187.
23. 901Giangaspero A, Sandri L, Tossi A: Amphipathic alpha helical antimicrobial902peptides.Eur J Biochem 2001,268:55895600.
24. 903Malina A, Shai Y: Conjugation of fatty acids with different lengths904modulates the antibacterial and antifungal activity of a cationic905biologically inactive peptide. Biochem J2005,390:695702.
25. 906Dewan PC, Anantharaman A, Chauhan VS, Sahal D: Antimicrobial action of907prototypic amphipathic cationic decapeptides and their branched908dimers.Biochemistry2009,48:56425657.
26. 909Trager W, Jensen JB: Human malaria parasites in continuous culture.910Science1976,193:673675.
27. 911MR4. http://www.mr4.org/MR4ReagentsSearch/Results.aspx?BEINum=MRA-
912819&Template=parasites.28. 913Lambros C, Vanderberg JP: Synchronization ofPlasmodium falciparum914erythrocytic stages in culture.J Parasitol1979,65:418420.
29. 915Rivadeneira EM, Wasserman M, Espinal CT: Separation and concentration916of schizonts ofPlasmodium falciparum by Percoll gradients. J Protozool9171983,30:367370.
30. 918Smilkstein M, Sriwilaijaroen N, Kelly JX, Wilairat P, Riscoe M: Simple and919inexpensive fluorescence-based technique for high-throughput antimalarial920drug screening.Antimicrob Agents Chemother2004,48:18031806.
31. 921Mosmann T:Rapid colorimetric assay for cellular growth and survival:922application to proliferation and cytotoxicity assays. J Immunol Meth 1983,92365:5563.
32. 924Yonath A:Polar bears, antibiotics, and the evolving ribosome (Nobel925Lecture).Angew Chem Int Ed Engl2010,49:43414354.
33. 926Matsuzaki K:Why and how are peptide-lipid interactions utilized for927self-defense? Magainins and tachyplesins as archetypes. Biochim Biophys928Acta1999,1462:110.
34. 929Shai Y:Mechanism of the binding, insertion and destabilization of930phospholipid bilayer membranes by alpha-helical antimicrobial and cell931non-selective membrane-lytic peptides. Biochim Biophys Acta1999,9321462:5570.
35. 933Yang L, Weiss TM, Lehrer RI, Huang HW:Crystallization of antimicrobial pores934in membranes: magainin and protegrin. Biophys J2000,79:20022009.
36. 935Ramagopal UA, Ramakumar S, Mathur P, Joshi R, Chauhan VS:936Dehydrophenylalanine zippers: strong helix-helix clamping through a937network of weak interactions. Protein Eng2002,15:331335.
37. 938Ramagopal UA, Ramakumar S, Sahal D, Chauhan VS: De novo design and939characterization of an apolar helical hairpin peptide at atomic resolution:940Compaction mediated by weak interactions. Proc Natl Acad Sci U S A9412001,98:870874.
38. 942Rudresh, Ramakumar S, Ramagopal UA, Inai Y, Goel S, Sahal D, Chauhan VS:943De novo design and characterization of a helical hairpin eicosapeptide;944emergence of an anion receptor in the linker region. Structure2004,
94512:389
396.39. 946Fichera ME, Roos DS:A plastid organelle as a drug target in
947apicomplexan parasites.Nature 1997,390:407409.40. 948Ralph SA, D'Ombrain MC, McFadden GI: The apicoplast as an antimalarial
949drug target.Drug Resist Updat2001,4:145151.41. 950Dahl EL, Rosenthal PJ: Multiple antibiotics exert delayed effects against
951thePlasmodium falciparum apicoplast.Antimicrob Agents Chemother2007,95251:34853490.
42. 953Hanssen E, McMillan PJ, Tilley L: Cellular architecture ofPlasmodium954falciparum-infected erythrocytes. Int J Parasitol2010,40:11271135.
43. 955Allred DR, Sterling CR, Morse PD 2nd: Increased fluidity ofPlasmodium956berghei-infected mouse red blood cell membranes detected by electron957spin resonance spectroscopy. Mol Biochem Parasitol1983,7:2739.
44. 958Howard RJ, Sawyer WH: Changes in the membrane microviscosity of959mouse red blood cells infected with Plasmodium berghei detected
Kaushiket al. Malaria Journal2012,11:256 Page 14 of 15
http://www.malariajournal.com/content/11/1/256
http://dx.doi.org/10.1038/news060116-13http://www.who.int/mediacentre/factsheets/fs094/en/index.htmlhttp://www.who.int/mediacentre/factsheets/fs094/en/index.htmlhttp://www.mr4.org/MR4ReagentsSearch/Results.aspx?BEINum=MRA-819&Template=parasiteshttp://www.mr4.org/MR4ReagentsSearch/Results.aspx?BEINum=MRA-819&Template=parasiteshttp://www.mr4.org/MR4ReagentsSearch/Results.aspx?BEINum=MRA-819&Template=parasiteshttp://www.mr4.org/MR4ReagentsSearch/Results.aspx?BEINum=MRA-819&Template=parasiteshttp://www.who.int/mediacentre/factsheets/fs094/en/index.htmlhttp://www.who.int/mediacentre/factsheets/fs094/en/index.htmlhttp://dx.doi.org/10.1038/news060116-137/27/2019 Anti-plasmodial Action of de Novo-Designed, Cationic, Lyisne Branched Amphipathic Helical Peptides
16/16
960 using n-(9-anthroyloxy) fatty acid fluorescent probes. Parasitology961 1980, 80 :331342.
45.962 Sherman IW, Greenan JR:Altered red cell membrane fluidity during963 schizogonic development of malarial parasites (Plasmodium falciparum964 and P. lophurae).Trans R Soc Trop Med Hyg 1984,78:641644.
46.965 Taraschi TF, Parashar A, Hooks M, Rubin H:Perturbation of red cell
966 membrane structure during intracellular maturation ofPlasmodium967 falciparum.Science1986,232:102104.
47.968 Vial HJ, Philippot JR, Wallach DF:A reevaluation of the status of969 cholesterol in erythrocytes infected by Plasmodium knowlesi and970 P. falciparum.Mol Biochem Parasitol1984,13:5365.
48.971 Vial HJ, Thuet MJ, Broussal JL, Philippot JR:Phospholipid biosynthesis by972 Plasmodium knowlesi-infected erythrocytes: the incorporation of973 phospohlipid precursors and the identification of previously undetected974 metabolic pathways.J Parasitol1982,68:379391.
49.975 Gupta CM, Mishra GC:Transbilayer phospholipid asymmetry in976 Plasmodium knowlesi-infected host cell membrane. Science 1981,977 212:10471049.
50.978 Joshi P, Dutta GP, Gupta CM:An intracellular simian malarial parasite979 (Plasmodium knowlesi)induces stage-dependent alterations in980 membrane phospholipid organization of its host erythrocyte.Biochem J981 1987,246:103108.
51.982 Schwartz RS, Olson JA, Raventos-Suarez C, Yee M, Heath RH, Lubin B, Nagel RL:983 Altered plasma membrane phospholipid organization in Plasmodium984 falciparum-infected human erythrocytes.Blood1987,69:401407.
52.985 Ginsburg H, Kutner S, Zangwil M, Cabantchik ZI:Selectivity properties of986 pores induced in host erythrocyte membrane byPlasmodium falciparum.987 Effect of parasite maturation.Biochim Biophys Acta1986,861:194196.
53.988 Kutner S, Ginsburg H, Cabantchik ZI:Permselectivity changes in malaria989 (Plasmodium falciparum) infected human red blood cell membranes.990 J Cell Physiol1983,114:245251.
54.991 Vial HJ, Ancelin ML:Malarial lipids. An overview. Subcell Biochem1992,992 18:259306.
55.993 Wender PA, Mitchell DJ, Pattabiraman K, Pelkey ET, Steinman L, Rothbard JB:994 The design, synthesis, and evaluation of molecules that enable or995 enhance cellular uptake: peptoid molecular transporters.Proc Natl Acad996 Sci U S A 2000,97:1300313008.
56.997 Rothbard JB, Jessop TC, Lewis RS, Murray BA, Wender PA:Role of998 membrane potential and hydrogen bonding in the mechanism of
999 translocation of guanidinium-rich peptides into cells. J Am Chem Soc1000 2004,126:95069507.
57.1001 Terrone D, Sang SL, Roudaia L, Silvius JR:Penetratin and related cell-1002 penetrating cationic peptides can translocate across lipid bilayers in the1003 presence of a transbilayer potential.Biochemistry2003,42:1378713799.
58.1004 Brown KL, Conboy JC:Electrostatic induction of lipid asymmetry. J Am1005 Chem Soc2011,133:87948797.
1006 doi:10.1186/1475-2875-11-2561007 Cite this article as:Kaushiket al.:Anti-plasmodial action ofde1008 novo-designed, cationic, lysine-branched, amphipathic, helical peptides.1009 Malaria Journal201211:256.
Submit your next manuscript to BioMed Centraland take full advantage of:
Convenient online submission
Thorough peer review
No space constraints or color figure charges
Immediate publication on acceptance
Inclusion in PubMed, CAS, Scopus and Google Scholar
Research which is freely available for redistribution
Submit your manuscript atwww.biomedcentral.com/submit
Kaushiket al. Malaria Journal2012,11:256 Page 15 of 15
http://www.malariajournal.com/content/11/1/256