Studies on the antileishmanial properties of the antimicrobial peptides temporin A, B and 1Sa

Article abstract of DOI:10.1002/psc.1398

Given the paucity and toxicity of available drugs for leishmaniasis, coupled with the advent of drug resistance, the discovery of new therapies for this neglected tropical disease is recognised as being of the utmost urgency. As such antimicrobial peptides (AMPs) have been proposed as promising compounds against the causative Leishmania species, insect vector-borne protozoan parasites. Here the AMP temporins A, B and 1Sa have been synthesised and screened for activity against Leishmania mexicana insect stage promastigotes and mammalian stage amastigotes, a significant cause of human cutaneous disease. In contrast to previous studies with other species the activity of these AMPs against L. mexicana amastigotes was low. This suggests that amastigotes from different Leishmania species display varying susceptibility to peptides from the temporin family, perhaps indicating differences in their surface structure, the proposed target of these AMPs. In contrast, insect stage L. mexicana promastigotes were sensitive to two of the screened temporins which clearly demonstrates the importance of screening AMPs against both forms of the parasite.

Full text of DOI:10.1002/psc.1398

Research Article
Received: 12 January 2011
Revised: 23 June 2011
Accepted: 24 June 2011
Published online in Wiley Online Library: 1 August 2011
(wileyonlinelibrary.com) DOI 10.1002/psc.1398
Studies on the antileishmanial proper-
ties of the antimicrobial peptides tem-
porin A, B and 1Sa
Frances L. Chadbourne,a Catriona Raleigh,a Hayder Z. Ali,a
Paul W. Dennya,b∗ and Steven L. Cobba∗
Given the paucity and toxicity of available drugs for leishmaniasis, coupled with the advent of drug resistance, the discovery of
new therapies for this neglected tropical disease is recognised as being of the utmost urgency. As such antimicrobial peptides
(AMPs) have been proposed as promising compounds against the causative Leishmania species, insect vector-borne protozoan
parasites. Here the AMP temporins A, B and 1Sa have been synthesised and screened for activity against Leishmania mexicana
insect stage promastigotes and mammalian stage amastigotes, a significant cause of human cutaneous disease. In contrast to
previous studies with other species the activity of these AMPs against L. mexicana amastigotes was low. This suggests that
amastigotes from different Leishmania species display varying susceptibility to peptides from the temporin family, perhaps
indicating differences in their surface structure, the proposed target of these AMPs. In contrast, insect stage L. mexicana
promastigotes were sensitive to two of the screened temporins which clearly demonstrates the importance of screening AMPs
c
against both forms of the parasite. Copyright ꢀ 2011 European Peptide Society and John Wiley & Sons, Ltd.
Supporting information may be found in the online version of this article
Keywords: Leishmania mexicana; cutaneous leishmaniasis; drug therapy; antimicrobial peptides; temporins
Introduction
administration [5]. Parasite resistance towards these second-line
drugs has not yet been conclusively confirmed in the field but
there are indications that it may only be a matter of time
[12]. Given the issues surrounding the use of the current first-
and second-line drugs to treat leishmaniasis there is clearly a
need to develop new and effective therapies to treat both CL
and VL.
In recent years one class of compounds that have been
investigated as antileishmanials is the antimicrobial peptides
(AMPs) [13,14]. AMPs isolated from a range of organisms have
been screened but to the best of our knowledge there are no
reports of any of these having been tested against the clinically
relevant amastigote form of Leishmania mexicana. L. mexicana
infection causes CL, which although non-fatal results in ulcerated
lesions that can cause severe disfigurement of skin tissue leading
to social exclusion in many affected communities. In addition,
CL lesions leave the patient at risk of severe secondary bacterial
Leishmaniasis is one of the World Health Organization’s 16
neglected tropical diseases and is endemic in over 80 countries
worldwide. It is caused by Leishmania species, insect vector-
borne protozoan parasites, and affects an estimated 12 million
people a year with a further 350 million people living at risk
of infection [1,2]. At the present time a vaccine to prevent
leishmaniasis is not available despite considerable research effort
in this area. Therefore, the treatment of leishmaniasis currently
relies entirely on a highly limited arsenal of chemotherapeutics.
The treatment of both visceral leishmaniasis (VL) and cutaneous
leishmaniasis (CL) largely rely on the pentavalent antimonials
such as sodium stibogluconate (Pentostam) and meglumine
antimoniate (Glucantime) [3,4]. These have been in clinical use
for over 70 years despite their associated problems, including
severe side effects such as renal failure and cardiotoxicity [5], and
the fact that they require parenteral administration [6]. However,
the most pressing issue surrounding the use of pentavalent
antimonials in the treatment of leishmaniasis comes from the
emergence of drug resistance [7]. Fortunately this has not, so
far, been widespread and remains isolated to the north Bihar
region of India where VL is endemic [8]. However, Leishmania
spp resistance to these drugs can be easily induced in the
laboratory [9]. Together these observations have led to concerns
that the antimonials may soon become redundant as effective
antileishmanials. Second-line drugs employed in the treatment
of both CL and VL include Amphotericin B (Fungizone) [10]
and the aromatic diamidine Pentamidine [11]. Both of these
drugs have been in clinical use for over 30 years and, like the
antimonials, induce severe side effects and require parenteral
∗
Correspondence to: Paul W. Denny, and Steven L. Cobb, Biophysical Sci-
ences Institute, Department of Chemistry and School of Biological and
Biomedical Sciences, Durham University, South Road, Durham DH1 3LE, UK.
E-mail: s.l.cobb@durham.ac.uk; p.w.denny@durham.ac.uk
a
BiophysicalSciencesInstitute,DepartmentofChemistryandSchoolofBiological
andBiomedicalSciences,DurhamUniversity,SouthRoad,DurhamDH13LE,UK
b
SchoolofMedicineandHealth,DurhamUniversity,Queen’sCampus,Stockton-
on-Tees TS17 6BH, UK
Abbreviations used: AMP, antimicrobial peptide; Amp, amphotericin B; CL,
cutaneous leishmaniasis; Pbf, 2,2,4,6,7-pentamethyldihydrobenzofurane; tBu,
tert-butyl; TIPS, triisopropylsilane; VL, visceral leishmaniasis. (All amino acid
symbols denote the L configuration.).
c
J. Pept. Sci. 2011; 17: 751–755
Copyright ꢀ 2011 European Peptide Society and John Wiley & Sons, Ltd.

CHADBOURNE ET AL.
infections. Given that several AMPs have been investigated as
potential treatments of other skin diseases [15] we sought to
explore any application that they may have in the treatment of CL.
Herein, we report the first examples of AMPs from the temporin
family being screened against L.mexicana, a causative agent of CL.
Table 1. Sequence, chemical and physical data for temporins
Rt
(HPLC)
Mr
Peptide
(Calc)a
Mr (obs)a,b
Temporin A
9.05
9.41
9.24
1395.9
1397.0
FLPLIGRVLSGIL-NH2
Temporin B
1390.9
1379.8
1413.8
1380.5
LLPIVGNLLKSLL-NH2
Temporin 1Sa
Materials and Methods
Reagents and Solvents
FLSGIVGMLGKLF-NH2
a The calculated (calc) and observed (obs) masses are monoisotopic.
b Mr (obs) are the observed protonated [M+H]+ or sodiated [M+Na]+
species as obtained by MALDI-TOF-MS.
Rink Amide AM resin (200–400 mesh, 0.62 mmol g−1 loading)
and all Fmoc-protected amino acids used were purchased from
Novabiochem, Merck (Nottingham, UK). PyBOP was purchased
from CEM (North Carolina, USA). HPLC-grade solvents were
obtained from Fischer Scientific (Loughborough, UK) and all
other reagents from Sigma-Aldrich (Gillingham, UK). Side chain
protecting groups utilised for the Fmoc amino acids were tBu for
Ser, Pbf for Arg, Boc for Lys and Trt for Asn.
Cytotoxicity Assay
Cytotoxicity analyses were performed in 96-well plates (Nunc,
Langenselbold, Germany) using Alamar Blue (Invitrogen, Paisley,
UK) with some modifications to the published protocol [17].
Briefly,followingoptimisationoftheassaysystem,100 µlofboth
promastigote and amastigote L. mexicana at 4 × 105 ml−1 were
incubated with compounds in triplicate (amphotericin B was used
as a positive control, and untreated parasites with vehicle (DMSO)
as a negative control) for 24 h before incubation with Alamar
Blue (Invitrogen) for 4 h prior to assessing cell viability using a
fluorescent plate reader (Biotek, Leeds, UK; 560EX nm/600EM nm).
To investigate the effects of serum on the efficacy of the temporins
the assay described above was modified. Briefly, promastigate and
amastigote L. mexicana were pre-incubated with the compounds
in 10 µl of serum-free media at 4 × 106 ml−1 for 1 h before the
addition of 90 µl of complete media [18]. All of the experiments
described above were carried out on a minimum of two separate
occasions to ensure a robust data set was collected.
Peptide Synthesis
Peptides were prepared via Fmoc SPPS using a CEM microwave
synthesiser. Peptides were synthesised on a 0.31 mmol scale
(500 mg of Rink Amide AM resin). Fmoc amino acids (4.0 equiv.
with respect to the resin) were coupled using PyBOP (3.9 equiv.
with respect to the resin), NMM (4.0 equiv. with respect to
the resin) and the CEM Microwave (10 min, 20 W, 75 ◦C). Fmoc
deprotection was carried out using piperidine 20%, DMF and the
CEM microwave (3 min, 20 W, 75 ◦C). Final peptide cleavage was
achieved using TFA : TIPS : H2O (9 ml:750 µl:750 µl) at 25 ◦C with
stirring for a minimum of 4 h.
Peptide Purification and Characterisation
Crude peptides were purified by semi-preparative RP-HPLC
(Waters, Milford, USA, Mass Directed Prep System instrument
fitted with a 3100 Mass detector and diode array detector) using a
Results and Discussion
Temporin A (1), temporin B (2) and temporin 1Sa (3) were chosen
for this study as they had previously been shown to have activity at
low micro-molar concentrations against other Leishmania species
[18,19]. Peptides 1–3 were prepared using a CEM microwave-
assisted Fmoc solid-phase procedure and their physical data are
presented in Table 1.
Given the ease of cell culture, AMPs have most commonly
been screened against insect stage, promastigote Leishmania
[13]. However, in order to fully assess the efficacy of any
compound, it must be assayed against pathogenic, mammalian
stage amastigotes. Therefore to facilitate comparative analyses of
the antileishmanial action of selected, synthesised temporins, it
was chosen to utilise Leishmania mexiciana, where axenic culture
of both lifecycle stages is long established [16].
19×100 mm Waters XBridge RP-column; flow rate = 17 ml min−1
;
linear gradient elution 10–90% of solvent B over 13.5 min
(A = 0.1% formic acid in H20, B = 0.1% formic acid in MeOH).
Peptides were characterised by analytical RP-HPLC (Waters Mass
Directed Prep System instrument fitted with a 3100 Mass detector
anddiodearraydetector)usinga4.6×100 mmWatersXBridgeRP-
column; flow rate = 1 ml min−1; linear gradient elution 10–90%
of solvent B over 16.5 min (A = 0.1% formic acid in H20, B = 0.1%
formicacidinMeOH).PeptideidentitieswereconfirmedbyMALDI-
TOF mass spectra analysis (Applied Biosystems, Carlsbad, USA,
Voyager-DE STR instrument operating in positive ion mode] using
an α-cyano-4-hydroxycinnamic acid matrix.
The Alamar Blue viability assay has previously been validated
for microtitre plate-based analyses of promastigote L. major [20];
and L. donovani, L. tropica [21] and L. mexicana [17] promastigotes
and amastigotes. In addition it has been utilised for screening
L. amazonensis, L. braziliensis and L. chagasi promastigotes
[22]. However, to facilitate comparison of the efficacy of the
synthesised AMPs against both promastigote and amastigote
axenic L. mexicana, the Alamar Blue assay was optimised to
allow both lifecycle stages to be screened under equivalent
conditions. To this end, and in the light of previous studies,
Leishmania Culture
Leishmania mexicana (MNYC/BZ/62/M379) parasites were main-
tained at 26 ◦C in Schneider’s Drosophila media (Sigma-Aldrich,
Gillingham, UK)supplementedwithheat-inactivatedfoetalbovine
sera (15% for promastigotes and 20% for amastigotes; Biosera
Ltd, East Sussex, UK). Promastigotes were transformed into ax-
enic amastigotes by a pH and temperature shift as previously
described [16]. Cells were counted using a Neubauer Improved
Haemocytometer.
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ANTILEISHMANIAL PROPERTIES OF TEMPORIN A, B AND 1SA
Figure 2. Using the Alamar Blue assay system, L. mexicana promastigote
(A) and amastigote (B) viability in the presence of various concentrations
of temporins A, B and 1Sa was determined with respect to untreated,
negative controls. Amphotericin B (Amp) was utilised as a positive control.
Data points represent the mean of 2 or 3 independent experiments
performed in triplicate. Standard deviation indicated.
linear relationship was apparent at higher cell concentrations
after 24 h incubation with the indicator, particularly with respect
to the promastigotes with the correlation breaking down at
more than 5 × 103 cells well−1 (Figure 1A). A similar pattern
has previously been noted with other Leishmania spp. [21]. To
ensure a direct correlation between readout and cell number in
the AMP screen of both lifecycle stages of L. mexicana, a starting
concentration of 4 × 105 ml−1 (4 × 104 cells well−1), followed
by incubation with Alamar Blue for 4 h, was employed in all
subsequent experiments.
Prepared peptides 1–3 were screened against both promastig-
ote and amastigote L. mexicana using the optimised Alamar
Blue assay. In the presence of serum only temporin A demon-
strated any significant antileishmanial activity against L. mexicana
promastigotes (Figure 2A, 57% inhibition at 100 µM). None of
the peptides (1–3) showed significant activity at the concentra-
tions tested against L. mexicana amastigotes (Figure 2B). Given
that the temporins investigated in this study (peptides1–3)
have previously been shown to be active other Leishmania
species [13], these results were unexpected. However, by pre-
incubating the parasites with the temporins studied in the
absence of serum the peptide activity profiles were altered. In
this assay, which reflects the conditions employed in previous
studies [18], temporins A and B demonstrated improved effi-
cacy against promastigote L. mexicana (Figure 3A; 63% inhibition
at 12.5 µM and 38% at 50 µM respectively). However, amastig-
ote forms remained largely resistant to these compounds with
only temporin A demonstrating any significant activity (Figure 3B;
only 23% inhibition at 12.5 µM but 98% at 100 µM). Notably,
Figure 1. Twofold serial dilutions starting at 4 × 105 ml−1 of Leishmania
mexicana promastigotes (A) and amastigotes (B) pre-incubated for 24 h
before the addition of Alamar Blue and further incubation for 4 h ( ) or
•
24 (ꢀ) h. Note the linear correlation between cell number and fluorescent
readout with a 4-h incubation (r2 = 0.999 in A and B). The lower signal
seen for amastigotes (B) reflects their relatively slow rate of replication [17].
All points in triplicate with standard deviation indicated. AFU – arbitrary
fluorescence units.
serial dilutions (starting at 4 × 104 cells well−1) of both lifecycle
stages were incubated for 24 h in 96-well plates at appropriate
temperatures before the addition of Alamar Blue at 10% v/v
for either 4 or 24 h and the subsequent assessment of cell
viability by fluorescent readout (Figure 1). The data clearly show
that a direct correlation of parasite numbers with readout was
apparent in the case of both lifecycle stages incubated for
4 h after the addition of Alamar Blue (r2 = 0.999 for both
promastigotes and amastigotes). However, deviation from this
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J. Pept. Sci. 2011; 17: 751–755 Copyright ꢀ 2011 European Peptide Society and John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/jpepsci

CHADBOURNE ET AL.
the temporin family indicating that broad-spectrum antileish-
manial AMPs may be challenging to develop. In addition, the
ability of AMPs to translocate the host membrane and reach
intra-macrophage amastigotes has not been widely studied
and remains unknown.
A
Acknowledgements
This work was supported by a Wolfson Research Institute
Small Grants Award (PWD and SLC), The Ramsay Memorial
Trust Fellowship (SLC), the Royal Society (RG090808) and the
Biotechnology and Biological Research Council (PWD, BB/
D52396X/1).
B
Supporting information
Supporting information for peptides prepared in Table 1 are
provided online.
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•
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