Investigating the anti-leishmanial effects of linear peptoids

Article abstract of DOI:10.1002/cmdc.201402416

Peptoids, a class of peptide mimetics, have emerged as promising anti-infective agents against a range of bacterial infections. Herein we present the first study of the antiparasitic and specifically the anti-leishmanial properties of linear peptoids. Peptoids were identified as having promising activity against Leishmania mexicana axenic amastigotes, a causative agent of cutaneous leishmaniasis.

Full text of DOI:10.1002/cmdc.201402416

DOI: 10.1002/cmdc.201402416
Communications
Investigating the Anti-leishmanial Effects of Linear
Peptoids
Gabriela A. Eggimann,[a] Hannah L. Bolt,[a] Paul W. Denny,*[a, b] and Steven L. Cobb*[a]
Peptoids, a class of peptide mimetics, have emerged as prom-
ising anti-infective agents against a range of bacterial infec-
tions. Herein we present the first study of the antiparasitic and
specifically the anti-leishmanial properties of linear peptoids.
Peptoids were identified as having promising activity against
Leishmania mexicana axenic amastigotes, a causative agent of
cutaneous leishmaniasis.
and development of safer, more effective treatments for VL. In
the 2012 London Declaration on Neglected Tropical Diseases,
public–private partnerships pledged to help control VL by
2020.[7] In contrast, CL, a disease that causes significant and
widespread medical and social problems with 1.2 million new
cases each year, remains relatively ignored.
Antimicrobial peptides (AMPs) have been proposed as one
potential solution to the development of new topical anti-
leishmanials to treat CL.[8] However, their inherent chemical
and biological instability presents a major hurdle, and only
a few AMPs are currently in clinical trials as antibacterial
agents.[9] In contrast, the peptide-mimicking peptoids offer
a significantly better opportunity for development from bench
to market.[10] Peptoids (or poly-N-substituted glycines) have
considerable potential for development as new topical anti-in-
fective agents, because they are cheaper to manufacture and
substantially more stable under physiological conditions.[11,12–15]
Their antibacterial activity against a range of Gram-positive
and Gram-negative bacteria (including MRSA), selectivity for
bacterial cells, and low hemolytic activity are similar to the
values reported for leading AMPs.[16,17]
The neglected tropical disease leishmaniasis is endemic in over
80 countries worldwide; 12 million people are infected, and
more than 350 million are at risk, the vast majority of whom
live in resource-poor settings with very limited access to health
care. Distinct insect-vector-borne Leishmania species can cause
either cutaneous leishmaniasis (CL), which leads to significant
scaring and mucosal damage (mucocutaneous leishmaniasis),
or visceral leishmaniasis (VL), which causes life-threatening
organ damage.[1,2] There is no vaccine for this serious wide-
spread disease, and current treatments largely rely on pentava-
lent antimonials such as sodium stibogluconate (Pentostam)
and meglumine antimoniate (Glucantime).[3,4] These have been
in clinical use for over 70 years, despite the associated severe
side effects such as cardiotoxicity and the need for parenteral
administration.
Herein we report the first ever screen of a linear peptoid li-
brary against Leishmania mexicana parasites, a causative spe-
cies of CL.[18] We identified active peptoids not only against
promastigotes (insect-stage parasites), but also against the
clinically more relevant axenic amastigotes (mammalian-stage
parasites). Our results provide a basis for further structure–ac-
tivity relationship (SAR) studies to investigate the mode of
action and toxicity of these new anti-leishmanial compounds.
All the peptoids in our library (Table 1) are designed around
the subunit NxNyNy, which is repeated either two, three, or
four times to give 6-, 9-, or 12-mers, respectively. For Ny we
chose either the chiral aromatic building block Nspe or the
achiral Nphe, as published work has shown that bulky aromatic
building blocks are needed in close proximity to induce a heli-
cal structure within the peptoid which can enhance antimicro-
bial activities.[14] For the positively charged building block Nx
we chose lysine-type amines with various side chain lengths
(Nah, NLys, and Nae) to study their influence on anti-leishmani-
al activity. It has been shown that this side chain length has an
influence on antimicrobial activity.[16]
The use of pentavalent antimonials in the treatment of leish-
maniasis for such a long time has led to the emergence of
drug resistance.[5] Therefore, second-line drugs such as ampho-
tericin B (Fungizone) and the aromatic diamidine pentamidine
have seen increasing use. Both have been in clinical use for
over 30 years and, like the antimonials, induce severe side ef-
fects and require parenteral administration. The newest and
only oral drug for VL, miltefosine, is also limited by its terato-
genicity.[6] Parasite resistance toward these alternative drugs
has not yet been conclusively confirmed in the field, but there
are indications that it may only be a matter of time.[5] Given
the issues surrounding the use of the current first- and
second-line drugs to treat leishmaniasis, there is clearly an
urgent need to develop new and effective therapies. Recently,
there has been an upsurge in interest regarding the discovery
[a] Dr. G. A. Eggimann, H. L. Bolt, Dr. P. W. Denny, Dr. S. L. Cobb
Biophysical Sciences Institute, Department of Chemistry
Durham University, South Road, Durham, DH1 3LE (UK)
E-mail: s.l.cobb@durham.ac.uk
The peptoids were synthesized manually on resin with the
sub-monomer method using a shaker (808C) and 15 min for
each coupling step. All peptoids were purified by RP-HPLC to
obtain a library of 18 compounds (see the Supporting Informa-
tion for details regarding synthesis and characterization).
Peptoids 1–18 were tested against L. mexciana promasti-
gotes and axenic amastigotes using a modified protocol that
was established in our research group.[8a] In brief, we incubated
[b] Dr. P. W. Denny
School of Medicine, Pharmacy and Health, Durham University
Queen’s Campus, Stockton-on-Tees, TS17 6BH (UK)
E-mail: p.w.denny@durham.ac.uk
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201402416.
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Table 1. Peptoid library synthesized on solid phase using the sub-monomer method: a) acylation step (bromoacetic acid and DIC in DMF); b) displacement
step (primary amine in DMF); c) TFA cleavage.
Nx
Ny
Peptoid
Sequence
Mr [Da]
No. cationic side chains
Nah
Nphe
1
2
3
(NahNpheNphe)4
(NahNpheNphe)3
(NahNpheNphe)2
1819.4
1368.8
918.2
+4
+3
+2
NLys
4
5
6
(NLysNpheNphe)4
(NLysNpheNphe)3
(NLysNpheNphe)2
1707.1
1284.6
862.1
+4
+3
+2
Nae
Nah
7
8
9
(NaeNpheNphe)4
(NaeNpheNphe)3
(NaeNpheNphe)2
1594.9
1200.4
806.0
+4
+3
+2
Nspe
10
11
12
(NahNspeNspe)4
(NahNspeNspe)3
(NahNspeNspe)2
1930.2
1452.9
974.3
+4
+3
+2
NLys
13
14
15
(NLysNspeNspe)4
(NLysNspeNspe)3
(NLysNspeNspe)2
1819.3
1368.8
918.2
+4
+3
+2
Nae
16
17
18
(NaeNspeNspe)4
(NaeNspeNspe)3
(NaeNspeNspe)2
1707.1
1284.6
862.1
+4
+3
+2
both promastigotes and axenic amastigotes for 1 h with the
appropriate concentrations of our compounds (2–100 mm). The
solutions were then diluted 10-fold, followed by a further incu-
bation for 24 h. Subsequently, alamarBlueꢁ was added, and
after 4 h the fluorescence was measured to determine the anti-
parasitic effect of the peptoids.
The results show that all short 6-mer peptoids 3, 6, 9, 12,
15, and 18 were completely inactive against both forms of the
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parasite, even at the highest concentration tested (Supporting
Information Figure S1). The longer 9-mer peptoids 11, 14, and
17 with the Nspe building block showed good activity against
the L. mexciana promastigotes (Figure 1a). Anti-leishmanial ac-
tivity increased as the side chain length of the positively
charged building block was shortened (Nae>NLys >Nah), with
peptoid 17 being the most active 9-mer in our library. In con-
trast, the 3-mer peptoids 2, 5, and 8 were not significantly
active against the promastigotes, indicating that the less steri-
cally hindered building block Nphe is not favored for antipara-
sitic activity. The 12-mer peptoids 1, 4, 7, 10, 13, and 16 were
all active, with the Nspe-containing peptoids again showing
significantly lower ED50 values than the Nphe analogues.
Again, as for the analogous 9-mer peptoids, a shorter side
chain (i.e., Nae) increased efficacy against promastigotes.
decrease cell viability by 40–100%, but at lower concentrations
activity was negligible. In contrast, peptoid 16 displayed
a good level of activity with a calculated ED50 value of 17 mm.
Notably, the ED50 value for peptoid 16 is similar to that report-
ed for AMPs against various amastigote-stage Leishmania
spp.[8b,19]
Because we used different amounts of serum in the promas-
tigote (15%) and amastigote (20%) media, we tested the pep-
toids in serum-free conditions in the first 1 h of incubation to
evaluate any effect of serum on activity. The results showed
that the serum had only a moderate effect on the antiparasitic
activity, so no inactive compounds became active, and the
ED50 values of the active compounds were only slightly de-
creased (see Supporting Information Figures S2 and S3). This is
contrast to previously reported work carried out on AMPs, in
which a clear and measurable difference between serum and
serum-free conditions was observed.[8a]
When we screened our library against the axenic amastigote
form of L. mexicana, several peptoids showed activity at
100 mm (Figure 1b). Peptoids 4, 7, 8, 10, and 17 were found to
Figure 1. Using the alamarBlueꢁ assay system, L. mexicana a) promastigote and b) amastigote viability in the presence of various concentrations of peptoids
(2–100 mm) was determined with respect to untreated, negative DMSO controls. Amphotericin B was used as a positive control. Nx=Nah, NLys or Nae. Data
points represent the mean of 2 or 3 independent experiments performed in triplicate, with standard deviation indicated; ED50 =50% effective dose. See Sup-
porting Information Figure S1 for a complete data set including 6-mer peptoids 3, 6, 9, 12, 15, and 18.
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tional Diagnostics). Peptoid syntheses were performed manually in
polypropylene syringes fitted with a polyethylene frit (20 mL, Craw-
ford Scientific) in a heated shaker (400 rpm, 808C). Preparative RP-
This first investigation of peptoids against the parasite
L. mexicana demonstrates that this class of peptide mimetics
has anti-leishmanial activity. We identified nine peptoids with
ED50 values in the low-micromolar range against the promasti-
gote form of the parasite and one peptoid with a low-micro-
molar ED50 value against the axenic amastigote form of the
parasite. Comparison of the sequences for the active peptoids
showed that both the overall length of the peptoid as well as
the nature of the individual building blocks influenced biologi-
cal activity. In general, as the peptoid sequence was length-
ened (i.e., moving from 6- to 12-mer), a lower ED50 value was
obtained. The chiral aromatic building block Nspe, which is
known to stabilize the formation of a helical secondary struc-
ture, was to found increase the antiparasitic effect relative to
the non-chiral Nphe. The additional methyl groups in the
Nspe-containing peptoids is also likely to increase the overall
hydrophobicity of the molecules, which may in turn help to
enhance the biological activity. In the side chains that bore the
positively charged amine group, we found that shorter chain
lengths gave rise to more active peptoids. Overall the combi-
nation of Nae (short side chain) and Nspe (chiral) building
blocks gave the most potent compound in the library, peptoid
16. This peptoid also demonstrated elevated cytotoxicity
against HepG2 cells, indicating that the increase in efficacy is
a generic effect. However, peptoid 16 was still significantly
more potent against L. mexicana amastigotes than the mam-
malian cell line (Supporting Information Table S5).
HPLC was performed with
a semi-preparative PerkinElmer
Series 200 LC pump fitted with a 785A UV/Vis detector using an
SB-Analytical ODH-S optimal column (250 mmꢂ10 mm, 5 mm; flow
rate 2 mLminÀ1). Peptoids were characterized by LC–MS (TQD mass
spectrometer and an Acquity UPLC from Waters) using an Acquity
UPLC BEH C8 1.7 mm (2.1 mmꢂ50 mm) column at a flow rate of
0.6 mLminÀ1 and a linear gradient of 5–95% solvent B over 3.8 min
(A=0.1% formic acid in H2O, B=0.1% formic acid in CH3CN). Pep-
toid identities were also confirmed by MALDI-TOF MS analysis (Au-
toflex II ToF/ToF mass spectrometer, Bruker Daltonik GmBH) operat-
ing in positive ion mode using an a-cyano-4-hydroxycinnamic acid
(CHCA) matrix. Data processing was done with MestReNova ver-
sion 8.1.
Synthesis and characterization of linear peptoids: Fmoc-protect-
ed Rink Amide resin (normally 100–300 mg, 0.1–0.3 mmol) was
swollen in DMF (1 h, room temperature) and deprotected with pi-
peridine (20% in DMF, v/v) for 30 min. After DMF washing, the
resin was treated with bromoacetic acid (8 equiv, 2m in DMF) and
DIC (8 equiv, 2m in DMF) for 15 min at 808C on a heated shaker
with a metal block (400 rpm, syringe was wrapped with tin foil for
better heat distribution). The resin was washed three times with
DMF (3ꢂ5 mL), before the desired amine sub-monomer was added
(4 equiv, 1m in DMF) and allowed to react for 15 min at 808C. The
resin was again washed three times with DMF (3ꢂ5 mL). The bro-
moacetylation and amine displacement steps were repeated until
the final sub-monomer had been added and the desired peptoid
sequence had been obtained. Cleavage was performed with TFA
(95%), TIPS (2.5%), and H2O (2.5%) for 90 min at room tempera-
ture. The cleavage cocktail was collected in a 50 mL falcon tube
and precipitated with 50 mL Et2O. The solution was centrifuged for
15 min at 4000 rpm (RCF 2570). Afterward, the ether phase was
decanted. The crude was dissolved in acidified H2O (with 0.1%
TFA) or a mixture of acidified H2O and CH3CN, and lyophilized. The
crude products were re-dissolved in acidified H2O (with 0.1% TFA)
or a mixture of acidified H2O and CH3CN, and purified by RP-HPLC.
A typical method used for the purification was 0–50% B over
60 min (A=0.1% TFA in 95% H2O and 5% CH3CN, B=0.1% TFA in
5% H2O and 95% CH3CN) at l 220 or 250 nm. Relevant fractions
were collected, lyophilized, and analyzed by LC–MS and MALDI-
TOF MS.
In conclusion, a library of 18 linear peptoids were prepared
and evaluated for anti-leishmanial activity. Twelve peptoids
from the library prepared showed activity against the promas-
tigote form of L. mexicana at ꢀ100 mm, with six (4, 10, 13, 14,
16, and 17) having ED50 values of <20 mm. Peptoid 16 was
found to be the most active, with an ED50 value of 7 mm. In
contrast, only seven peptoids from the library showed activity
against L. mexicana axenic amastigotes at ꢀ100 mm. Only pep-
toid 16 was found to have an ED50 value of <20 mm (17 mm).
Our investigation also demonstrated a clear difference in the
sensitivity of the two different parasite forms of L. mexciana to
peptoids, with amastigotes being significantly more resistant.
This difference is also observed for AMPs,[8,20] and it suggests
that the different cellular surfaces of promastigotes and amas-
tigotes[21] influences sensitivity to peptoids and peptides. Lead-
ing on from this, it is likely that the peptoids screened in this
study target the cellular membrane of the parasites to affect
their biological activity.
Cell culture of L. mexicana (M379) promastigotes and amasti-
gotes: Leishmania mexicana (MNYC/BZ/62/M379) promastigote
parasites were maintained at 268C in Schneider’s Insect medium
(Sigma–Aldrich) supplemented with heat-inactivated fetal bovine
serum (FBS, 15%; Biosera Ltd.). Cells were counted using a Neuba-
uer Improved Hemocytometer. Promastigotes were transformed
into axenic amastigotes by a pH and temperature shift as previous-
ly described:[21] In brief, a culture of promastigotes in the late log
phase was transferred into Schneider’s Insect medium supplement-
ed with 20% heat-inactivated FBS (pH 5.5) at 5ꢂ105 parasites per
mL. After six days, the parasites were in the metacyclic stage and
used for transformation into amastigote forms by transfer in the
same medium at 328C at 5ꢂ105 parasites per mL. After an addi-
tional 5–7 days, the parasites were in the amastigote stage.
Experimental Section
Materials and reagents: Abbreviations for reagents are as follows:
tert-butoxycarbonyl (Boc); 9-fluorenylmethoxylcarbonyl (Fmoc); tri-
fluoroacetic acid (TFA); triisopropylsilyl (TIPS); N,N-dimethylforma-
mide (DMF); N,N-diisopropylcarbodiimide (DIC); dimethylsulfoxide
(DMSO). Solvents and reagents were purchased from commercial
sources and used without further purification unless otherwise
Cytotoxicity assays with L. mexicana promastigotes and amasti-
gotes: Cytotoxicity analyses were performed in 96-well plates (flat-
bottom, Costar, Fisher Scientific) using alamarBlueꢁ (Life Technolo-
gies) for cell viability detection as previously described.[8] L. mexica-
na promastigotes and amastigotes were pre-incubated with the
noted. Rink amide resin (typical loading level 0.6–0.8 mmolgÀ1
)
was purchased from Merck Biosciences. Bromoacetic acid, TFA, DIC,
and the amine building blocks were purchased from Sigma–Aldrich
or TCI Europe. DMF was purchased from AGTC Bioproducts (Na-
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compounds in triplicate (5 mm stock solutions in DMSO; amphoter-
icin B was used as a positive control; untreated parasites with
DMSO as a negative control) in 50 mL of the corresponding media
at 4ꢂ106 mLÀ1 for 1 h. Afterward, 40 mL were removed from each
well before the addition of 90 mL of the corresponding media, fol-
lowed by incubation for 24 h at appropriate temperature. Then,
10 mL alamarBlueꢁ solution was added to each well before a 4 h in-
cubation at the appropriate temperature prior to assessing cell via-
bility using
a fluorescent plate reader (Biotek; lex 560 nm,
lem 600 nm). To investigate the effects of serum on the efficacy of
the peptoids, the assay described above was modified using
serum-free medium for the pre-incubation time. For these assays,
the parasites were washed three times in serum-free medium
before adding them to the compound solutions. All of the experi-
ments described above were carried out on a minimum of two
separate occasions to ensure a robust data set was collected.
[15] J. A. Patch, A. E. Barron, J. Am. Chem. Soc. 2003, 125, 12092.
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Acknowledgements
[17] R. Kapoor, M. W. Wadman, M. T. Dohm, A. M. Czyzewski, A. M. Spor-
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[18] To the best of our knowledge, only one other investigation into the an-
tiparasitic properties of linear peptoids has been reported: C. Chan, H,
Yin, J. H. McKie, A. H. Fairlamb, K. T. Douglas, Amino Acids 2002, 22,
297–308. Notably, this work by Chan et al. details peptoid inhibitory ac-
tivity against a specific enzyme target, trypanothione reductase from
Trypanosoma cruzi.
We thank the Engineering and Physical Sciences Research Coun-
cil, UK (H.L.B.) and the Swiss National Science Foundation (G.A.E.)
for financial support.
Keywords: anti-leishmanial agents · antiparasitic agents ·
Leishmania · peptoids
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Published online on && &&, 0000
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G. A. Eggimann, H. L. Bolt, P. W. Denny,*
S. L. Cobb*
L. mex. meets its match: Peptoids—
a class of peptide mimetics—have dem-
onstrated their activities against a range
of bacterial infections. In this work, we
studied the anti-leishmanial properties
of linear peptoids, which were observed
to have promising activity against Leish-
mania mexicana axenic amastigotes,
a causative agent of cutaneous leishma-
niasis.
&& – &&
Investigating the Anti-leishmanial
Effects of Linear Peptoids
&
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