Antiplasmodial activity of short peptide-based compounds

Article abstract of DOI:10.1039/c5ra00779h

Three series of short peptide-based compounds were synthesized, which upon evaluation against chloroquine-sensitive (D6) and chloroquine-resistant (W2) strains of Plasmodium falciparum in vitro, produced IC₅₀ values ranging between 1.4-4.7 μg mL^-1. Importantly, higher antimalarial activity against the drug-resistant strain of P. falciparum (W2) was observed for the tested peptides, indicating their potential in the treatment of drug-resistant malaria parasites. The lack of cytotoxicity in all tested peptides provides evidence of their safety profile. The selected peptides were evaluated in an enzymatic inhibitory assay against plasmepsin II, a potential target for antiplasmodial activity, also indicated from the results of the molecular docking studies.

Full text of DOI:10.1039/c5ra00779h

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Antiplasmodial activity of short peptide-based
compounds†
Cite this: RSC Adv., 2015, 5, 22674
Amit Mahindra,a Rahul P. Gangwal,b Sunil Bansal,a Nathan E. Goldfarb,c Ben M. Dunn,c
b
a
*
Abhay T. Sangamwar and Rahul Jain
Three series of short peptide-based compounds were synthesized, which upon evaluation against
chloroquine-sensitive (D6) and chloroquine-resistant (W2) strains of Plasmodium falciparum in vitro,
produced IC50 values ranging between 1.4–4.7 mg mLꢀ1. Importantly, higher antimalarial activity against
the drug-resistant strain of P. falciparum (W2) was observed for the tested peptides, indicating their
potential in the treatment of drug-resistant malaria parasites. The lack of cytotoxicity in all tested
peptides provides evidence of their safety profile. The selected peptides were evaluated in an enzymatic
inhibitory assay against plasmepsin II, a potential target for antiplasmodial activity, also indicated from the
results of the molecular docking studies.
Received 14th January 2015
Accepted 12th February 2015
DOI: 10.1039/c5ra00779h
www.rsc.org/advances
membrane organelles with an internal pH between 4 and 5.4.
Four of the ten aspartic proteases found in the genome, have
Introduction
been localized to the acidic food vacuole of the parasite, namely
plasmepsin I, II, IV, and HAP, a histo-aspartic protease.6 Plm I
and Plm II make the rst strategic cleavage of hemoglobin
between Phe33 and Leu34 of the R-chain, resulting in protein
unfolding and release of the heme moiety. Subsequent degra-
dation steps are catalyzed by the cysteine protease falcipain, the
metalloprotease falcilysin, and cytoplasmic aminopeptidases
(Fig. 1). The amino acids derived from hemoglobin degradation
are utilized by the parasite as an energy source.7 In addition, the
clearance of hemoglobin from the erythrocyte provides space
for the growing parasite.
With approximately 219 million cases and 660 000 attributed
deaths reported globally in 2013, malaria is one of the most
severe infectious diseases in the world.1 In Africa alone, more
than one million children under the age of ve die from malaria
each year, and it remains inextricably linked with the poverty.
The unavailability of a vaccine and the spread of drug resistance
over the past 15–20 years have led to a dramatic decline in the
efficacy of the most affordable drugs.2 Human infection can be
caused by four major species of the malaria parasite, i.e., Plas-
modium falciparum, P. vivax, P. malariae, and P. ovale, of which
P. falciparum is responsible for more than 95% of malaria-
related morbidity and mortality.3 P. falciparum, the most
lethal of the four parasites infecting humans, is increasingly
becoming resistant to many of the current frontline antima-
larial drugs including, chloroquine, quinine, artemisinin, and
sulfadoxime/pyrimethamine.4 This has been recognized as a
major health concern, highlighting the urgent need for new
treatment paradigms and for new efficacious drugs to combat
this disease.
The inhibition of any of these enzymes leads to the starva-
tion of the parasite and has been proposed as a viable strategy
for drug development.8 Plm II is the most thoroughly studied
The life cycle of Plasmodium parasite starts with the invasion
into human erythrocytes and consumes up to 75% of the
hemoglobin present.5 The digestive vacuoles are acidic single-
aDepartment of Medicinal Chemistry, National Institute of Pharmaceutical Education
and Research, Sector 67 S.A.S Nagar-160062, Punjab, India. E-mail: rahuljain@niper.
ac.in; Fax: +91-172-2214692
bDepartment of Pharmacoinformatics, National Institute of Pharmaceutical Education
and Research, Sector 67 S.A.S Nagar-160062, Punjab, India
cDepartment of Biochemistry and Molecular Biology, University of Florida, Gainesville,
32611, USA
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c5ra00779h
Fig. 1 Degradation of hemoglobin by plasmepsin and associated
proteases.
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Fig. 2 Known antiplasmodial peptides.
Fig. 4 Loading of various amino acids on to the 2-chlorotrityl chloride
resin under optimized conditions.
molecular docking studies against Plm II of three series of short
peptide-based inhibitors.
Results and discussion
The basis of this study was to develop new compounds acting
through distinct mechanisms during both the liver stage and
the blood stage of the life cycle of the parasite.12 In search of
peptide-based inhibitors of P. falciparum, we came across a
number of natural and synthetic peptides having diverse
sequences. Two long linear peptides psalmopeotoxin I (33
amino acids) and psalmopeotoxin II (27 amino acids) were
isolated from the venom of tarantula having IC50 values of 1.59
mM and 1.15 mM against P. falciparum.13 Antimicrobial peptide
dermaseptin S4 derivatives were also identied for anti-
plasmodial activity. In a study, it has been observed that the
truncated dermaseptin S4 from several portions, and their
shorter sequences retained the antiplasmodial activity.14
Fig. 3 Structure of the lead compounds.
enzyme among the aspartyl proteases of Plasmodium.9 In 1996,
the crystal structure of plasmepsin II complexed with pepstatin
A was published.10 It is made up of 329 amino acids folded into
two topologically similar N- and C-terminal domains and may
be a suitable molecular target for future antimalarial drugs. In
the light of availability of the crystal structure of Plm II, a
number of inhibitors have been reported in the past.11
In this paper we report the synthesis, biological evaluation
against P. falciparum growth, enzymatic inhibition assay and
Several natural peptides were isolated from different
resources and exhibit activity against P. falciparum with a
Table 1 Optimization of loading of resin under various sets of reaction conditionsa
Loading
2-CTCa
Entry no.
Base
Solvent
Equiv. of base
Time (h)
Wangb
1
2
3
4
5
6
7
8
DMAP
TEA
DBU
DCM
DCM
DCM
DCM
DCM
1,4-Dioxane
THF
DCE
DMF
NMP
DMF
3
3
3
3
3
3
3
3
3
3
3
6–9
6–9
6–9
6–9
6–9
6–9
6–9
6–9
6–9
6–9
6–9
0.47
0.24
0.31
0.56
0.38
0.42
0.43
0.54
0.69
0.43
0.78
0.40
0.28
0.25
0.48
0.30
0.36
0.34
0.44
0.53
0.35
0.59
DIEA
Collidine
DIEA
DIEA
DIEA
DIEA
DIEA
DIEA
9
10
11
a
Reaction conditions: Fmoc-Orn(Boc)-OH (3 equiv.), base (3 equiv.), solvent (2 mL). b Fmoc-Orn(Boc)-OH (3 equiv.), TBTU (3 equiv.), solvent (2 mL).
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Scheme 1 Generalized scheme for solid phase peptide synthesis.
range of IC50 values of 4–9 mM (Fig. 2), but produced cyto- moiety is observed that plausibly coordinates to the cata-
toxicity in Vero cells.15–19 As shown in Fig. 2, tetrapeptide lytic dyad of an aspartic protease as described in the earlier
hirsutellic acid A15 having amino acids isoleucine (I, Ile), reports.20
leucine (L, Leu) and N-methylphenylalanine (N-Me-Phe)
In a recent study, E. Candol and co-workers have identied
exhibit IC50 value of 8.0 mM against P. falciparum, and was a small peptide fragment Leu-Ser-Phe-Arg from various chro-
non-cytotoxic to Vero cells at a concentration of 95 mM. Other mogranins sequences.21 This small peptide inhibits 50% growth
antiplasmodial peptides isolated from natural resources are of P. falciparum strain 3D7 at 20 mM and satises the minimum
dragonamide A16 and gallinamide 1,17 that exhibit IC50 and pharmacophoric requirement for activity having cationic amino
cytotoxicity values of 7.7 mM and 67.8 mM (dragonamide), and acid Arg, hydrophobic amino acids Leu and Phe, and also the
8.4 mM and 10.4 mM (gallinamide). Synthetic peptides were hydroxyl group-containing amino acid serine (Fig. 3). In another
also reported in the literature having potent antimalarial independent study, two tetrapeptides having sequence Phe-Orn-
activity with best exhibiting IC50 value of 0.56 nM.18,19 A key Phe-Orn and Lys-Phe-Phe-Orn showed antimalarial activity with
structural feature in these sequences is the presence of IC50 values of 3.31 and 2.57 mM, respectively (Fig. 3).22 Herein,
hydrophobic amino acids such as Phe, Leu, Valine (V, Val) compounds were designed on the concept of bioisosteric
and Ile. Also the presence of a hydroxyl or hydroxyl-like replacement, which is an established strategy to have been
extensively used to optimize lead compounds in the peptide-
based drug discovery.23,24
Chemistry
To synthesize desired peptides, we rst optimized the loading of
resin in our laboratory.25,26 The optimization of the loading of
resin for different amino acids (AAs) was performed under a
variety of reaction conditions. The loading capacity was deter-
mined by standard protocol assay.27
In order to optimize the loading on 2-chlorotrityl chloride (2-
CTC) and Wang resins, rst we screened a number of homog-
enous bases. As shown in Table 1, the most promising results
were obtained with N,N-diisopropylethylamine (DIEA) in DCM
(dichloromethane) (entry no. 4). In case of DMAP (4-dimethyl-
aminopyridine) (entry no. 1), the loading is somewhat prom-
ising, whereas with other bases such as TEA (triethylamine),
Fig. 5 Representative example of peptide modification at various
positions.
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Table 2 In vitro antimalarial activities of peptides (series 1)a
P. falciparum (D6)
P. falciparum (W2)
Peptide
Substitution
Sequence
IC50 (mg mLꢀ1
)
SIb
IC50 (mg mLꢀ1
)
SIb
Modication at R1
7
6
8
9
10
11
12
13
14
Leu
Tle
Ile
Val
L-S-F-R
Tle-S-F-R
I-S-F-R
NA
3.6
NA
NA
4.2
4.0
4.0
3.0
NA
ꢀ
NA
3.2
NA
NA
4.0
3.8
3.6
3.0
NA
ꢀ
>2.7
ꢀ
>3.1
ꢀ
V-S-F-R
ꢀ
ꢀ
Nle
Nle-S-F-R
hLeu-S-F-R
Nval-S-F-R
Aib-S-F-R
D-L-S-F-R
>2.3
>2.5
>2.5
>3.3
ꢀ
>2.5
>2.6
>2.7
>3.3
ꢀ
homoLeu
Nval
Aib
D-Leu
Modication at R2
15
16
17
18
19
Tyr
Thr
homoSer
N-Me-Ser
D-Ser
L-Y-F-R
L-T-F-R
L-hSER-F-R
L-NMeSer-F-R
L-D-S-F-R
NA
NA
NA
NA
2.1
ꢀ
NA
NA
NA
NA
1.8
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
>4.7
>5.5
Modication at R3
20
21
22
23
24
25
26
27
28
2-Nal
L-S-2-Nal-R
L-S-F(4-Me)-R
L-S-F(4-Br)-R
L-S-F(4-Cl)-R
L-S-F(4-NH2)-R
L-S-F(4-t-Bu)-R
L-S-hPhe-R
L-S-F-R
2.4
NA
3.0
2.4
2.0
3.8
NA
3.2
2.1
>4.1
ꢀ
1.8
NA
2.0
1.7
1.6
3.2
NA
3.0
1.8
>5.5
ꢀ
Phe(4-Me)
Phe(4-Br)
Phe(4-Cl)
Phe(4-NH2)
Phe(4-t-Bu)
homoPhe
Cha
>3.3
>4.1
>5
>5
>5.8
>6.2
>3.1
ꢀ
>2.6
ꢀ
>3.1
>4.7
>3.3
>5.5
D-Phe
L-S-D-F-R
Modication at R4
29
30
31
32
33
34
35
36
Cit
Orn
Dab
His(1-Bzl)
Lys
His
D-Arg
D-Arg*
L-S-F-Cit
NA
NA
NA
NA
2.0
NA
NA
1.9
ꢀ
NA
NA
NA
NA
1.7
NA
NA
1.7
ꢀ
L-S-F-Orn
L-S-F-Dab
L-S-F-H(1-Bzl)
L-S-F-K
L-S-F-H
L-S-F-D-R
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
>5
ꢀ
>5.8
ꢀ
ꢀ
ꢀ
D-L-D-S-D-F-D-R
>5.2
>5.8
a
IC50 are the sample concentration that kills 50% cells compared to vehicle control; NC, not cytotoxic up to (10 mg mLꢀ1); NA, not active; “ꢀ”, not
calculated. Chloroquine: IC50 ¼ 0.014 mg mLꢀ1, SI ¼ 714 (D6 clone); IC50 ¼ 0.1 mg mLꢀ1, SI ¼ 100 (W2 clone); artemisinin: IC50 ¼ 0.015 mg mLꢀ1
,
SI ¼ 666 (D6 clone); IC50 ¼ 0.009 mg mLꢀ1, SI ¼ 1111 (W2 clone). b Selectivity index (SI) is the ratio of IC50 in Vero cells to IC50 in P. falciparum (D6 or
W2). * Containing all D-amino acids.
DBU (1,8-diazabicyclo[5.4.0]undec-7-ene) and collidine, the optimal conditions were dened for the cross-linking of the
loading was low (entry nos 2, 3 and 5). Aer optimization of the resin into amino acids, desired amino acids was preloaded on
base, a number of non-polar solvents like THF (tetrahydro- the solid support (Fig. 4). The peptides for the desired study
furan), DCE (1,2-dichloroethene) and polar solvents such as were synthesized using the Fmoc protocol of the solid phase
NMP (N-methyl-2-pyrrolidone) and DMF (dimethylformamide) peptide synthesis (SPPS) on a fully automated peptide synthe-
were screened. DMF (entry no. 9) appears to be best among all sizer (Aapptec, Focus XC) as shown in the Scheme 1.
the screened solvents. Aer optimization of base and solvent,
A library of peptides was synthesized based on lead peptides
we performed the double coupling of amino acids in the pres- I, II and III. We concentrated our efforts on a systematic amino
ence of DIEA as a base, and DMF as solvent. We found that this acid replacement strategy without increasing the peptide
condition gives the best loading results (entry no. 11), among all backbone length. The rst site examined was the N-terminus
the experiments performed.
amino acid in the lead peptide I. Leucine was replaced with
The extent of racemization during the optimized loading other hydrophobic and sterically hindered amino acids at the R1
procedure was determined by preparing the dipeptides, which position to see the effect of its replacement on the antimalarial
upon HPLC analysis was found to be less than 0.1%. Aer activity of the peptides keeping the other three amino acids
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Table 3 In vitro antimalarial activities of peptides (series 2)a
P. falciparum (D6)
P. falciparum (W2)
Peptide
Substitution
Sequence
IC50 (mg mLꢀ1
)
SIb
IC50 (mg mLꢀ1
)
SIb
Modication at R1
37
38
39
40
41
42
43
Lys
K-F-F-O
2.0
NA
NA
2.6
NA
NA
NA
>5
ꢀ
1.6
NA
NA
1.8
NA
NA
NA
>6.2
ꢀ
Orn
Dab
Arg
Cit
His
D-Lys
O-F-F-O
Dab-F-F-O
R-F-F-O
Cit-F-F-O
H-F-F-O
D-K-F-F-O
ꢀ
ꢀ
>3.8
ꢀ
>5.5
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
Modication at R2
44
45
46
47
48
49
Phe(4-NH2)
Cha
Phe(4-Cl)
Phe(4-t-Bu)
homoPhe
D-Phe
K-F(4-NH2)-F-O
K-Cha-F-O
K-F(4-Cl)-F-O
K-F(4-t-Bu)-F-O
K-hPhe-F-O
K-D-F-F-O
1.6
NA
1.6
NA
NA
NA
>6.2
ꢀ
1.4
NA
1.4
NA
NA
NA
>7.1
ꢀ
>6.2
ꢀ
>7.1
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
Modication at R3
50
51
52
53
54
55
Phe(4-NH2)
Cha
Phe(4-Cl)
Phe(4-t-Bu)
homoPhe
D-Phe
K-F-F(4-NH2)-O
K-F-Cha-O
K-F-F(4-Cl)-O
K-F-F(4-t-Bu)-O
K-F-hPhe-O
K-F-D-F-O
NA
NA
NA
2.0
NA
NA
ꢀ
ꢀ
ꢀ
>5
ꢀ
ꢀ
NA
NA
NA
1.6
NA
NA
ꢀ
ꢀ
ꢀ
>6.2
ꢀ
ꢀ
Modication at R4
56
57
58
59
60
61
62
Arg
His
Cit
Dab
Lys
K-F-F-R
K-F-F-H
2.0
3.2
NA
2.6
2.6
NA
NA
>5
1.6
3.0
NA
1.8
1.8
NA
NA
>6.2
>3.1
ꢀ
>3.1
ꢀ
K-F-F-Cit
K-F-F-Dab
K-F-F-K
K-F-F-D-O
D-K-D-F-D-F-D-O
>3.8
>3.8
ꢀ
>5.5
>5.5
ꢀ
D-Orn
D-Orn*
ꢀ
ꢀ
a
IC50 are the sample concentration that kills 50% cells compared to vehicle control; NC, not cytotoxic up to (10 mg mLꢀ1); NA, not active; “ꢀ”, not
calculated. Chloroquine: IC50 ¼ 0.014 mg mLꢀ1, SI ¼ 714 (D6 clone); IC50 ¼ 0.1 mg mLꢀ1, SI ¼ 100 (W2 clone); artemisinin: IC50 ¼ 0.015 mg mLꢀ1
,
SI ¼ 666 (D6 clone); IC50 ¼ 0.009 mg mLꢀ1, SI ¼ 1111 (W2 clone). b Selectivity index (SI) is the ratio of IC50 in Vero cells to IC50 in P. falciparum (D6 or
W2). * Containing all D-amino acids.
intact (Fig. 5). Next, to explore the importance of polar amino
acid serine (Ser) at the position 2; we replaced it with other position, peptide 13 (R1 ¼ Aib) produced promising activity
hydrophilic amino acids (containing a hydroxyl group) at the R2 against D6 and W2 strains with IC50 values of 3.0 mg mLꢀ1
Among peptides 6–14, which represent replacement at the R1
.
position in lead compound I. Similarly, we systematically While analogs 6 (R1 ¼ Tle), 10 (R1 ¼ Nle), 11 (R1 ¼ homoLeu)
replaced the phenylalanine (Phe) residue at the position 3 and and 12 (R1 ¼ Nval) showed activity against D6 and W2 strains
the C-terminus amino acid arginine in the lead peptide I with its with IC50 values of 3.6 and 3.2 mg mLꢀ1, 4.2 and 4.0 mg mLꢀ1, 4.0
bioisosteric counterparts. Similar strategy was adopted to and 3.8 mg mLꢀ1, 4.0 and 3.6 mg mLꢀ1, respectively. The peptide
synthesize peptides of series 2 and 3.
analog 14 containing all D-amino acids was inactive at the
highest tested concentration of 10 mg mLꢀ1
Peptides 15–19 represents replacement at the R2 position in
lead peptide I. Of these, peptide 19 (R2 D-Ser) exhibited
.
Antiplasmodial activity
¼
encouraging activity against D6 and W2 strains with IC50 values
of 2.1 and 1.8 mg mLꢀ1, respectively. Preliminary structure
activity observations indicate that only Ser containing peptide
has demonstrated antiplasmodial activity from this series of
compounds. In general, replacement of Ser residue at the 2
position in the lead compound I results in inactive peptides.
Peptides 20–28, represent replacement of Phe residue at the
All synthesized peptides were tested in vitro for antimalarial
activity against chloroquine sensitive (D6) and resistant P. fal-
ciparum strains (W2) and results are shown in Tables 2–4. In
vitro antimalarial activity was determined on the basis of plas-
modial LDH activity, and expressed as IC50 values versus
chloroquine-sensitive (D6) and chloroquine-resistant (W2)
strains of P. falciparum.28,29
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Table 4 In vitro antimalarial activities of peptides (series 3)a
P. falciparum (D6)
P. falciparum (W2)
Peptide
Substitution
Sequence
IC50 (mg mLꢀ1
)
SIb
IC50 (mg mLꢀ1
)
SIb
Modication at R1
63
64
65
66
67
68
69
Phe
F-O-F-O
4.0
2.0
2.6
4.0
NA
NA
NA
>2.5
>5
4.0
1.6
1.8
4.0
NA
NA
NA
>2.5
>6.2
>5.5
>2.5
ꢀ
Phe(4-Cl)
Phe(4-NH2)
Phe(4-t-Bu)
Cha
homoPhe
D-Phe
F(4-Cl)-O-F-O
F(4-NH2)-O-F-O
F(4-t-Bu)-O-F-O
Cha-O-F-O
hPhe-O-F-O
D-F-O-F-O
>3.8
>2.5
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
Modication at R2
70
71
72
73
74
75
Lys
His
Arg
Cit
Dab
D-Orn
K-O-F-O
H-O-F-O
R-O-F-O
Cit-O-F-O
Dab-O-F-O
F-D-O-F-O
NA
4.7
NA
NA
3.2
NA
ꢀ
NA
4.2
NA
NA
3.0
NA
ꢀ
>2.1
ꢀ
>2.1
ꢀ
ꢀ
ꢀ
>3.1
ꢀ
>3.3
ꢀ
Modication at R3
76
77
78
79
80
81
Phe(4-Cl)
Phe(4-NH2)
Phe(4-t-Bu)
Cha
homoPhe
D-Phe
F-O-F(4-Cl)-O
F-O-F(4-NH2)-O
F-O-F(4-t-Bu)-O
F-O-Cha-O
F-O-hPhe-O
F-O-D-F-O
2.0
2.6
1.6
NA
NA
NA
>5
1.6
1.8
1.4
NA
NA
NA
>6.2
>5.5
>7.1
ꢀ
>3.8
>6.2
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
Modication at R4
82
83
84
85
86
87
88
Arg
His
Lys
Dab
Cit
F-O-F-R
F-O-F-H
F-O-F-K
F-O-F-Dab
F-O-F-Cit
F-O-F-D-O
D-F-D-O-D-F-D-O
1.6
NA
2.6
2.6
NA
NA
NA
>6.2
ꢀ
1.4
NA
1.8
1.8
NA
NA
NA
>7.1
ꢀ
>3.8
>3.8
ꢀ
>5.5
>5.5
ꢀ
D-Orn
D-Orn*
ꢀ
ꢀ
ꢀ
ꢀ
a
IC50 are the sample concentration that kills 50% cells compared to vehicle control; NC, not cytotoxic up to (10 mg mLꢀ1); NA, not active; “ꢀ”, not
calculated. Chloroquine: IC50 ¼ 0.014 mg mLꢀ1, SI ¼ 714 (D6 clone); IC50 ¼ 0.1 mg mLꢀ1, SI ¼ 100 (W2 clone); artemisinin: IC50 ¼ 0.015 mg mLꢀ1
,
SI ¼ 666 (D6 clone); IC50 ¼ 0.009 mg mLꢀ1, SI ¼ 1111 (W2 clone). b Selectivity index (SI) is the ratio of IC50 in Vero cells to IC50 in P. falciparum (D6 or
W2). * Containing all D-amino acids.
3 position in the lead peptide with its aromatic hydrophobic 23 [R3 ¼ Phe(4-Cl)] and 28 (R3 ¼ D-Phe) also showed encour-
counterparts. Analog 24 [R3 ¼ Phe(4-NH2)] produced promising aging activities against D6 and W2 strains by exhibiting IC50
activity against D6 and W2 strains with IC50 values of 2.0 and values of 2.4 and 1.8 mg mLꢀ1, 2.4 and 1.7 mg mLꢀ1 and 2.1 and
1.6 mg mLꢀ1, respectively. While, analogs 20 (R3 ¼ 2-Nal), 1.8 mg mLꢀ1, respectively.
The analogs 22 [R3 ¼ Phe(4-Br)], 25 [R3 ¼ Phe(4-t-Bu)] and 27
(R3 ¼ Cha) also showed comparable activities against D6 and
W2 strains with IC50 values of 3.0 and 2.0 mg mLꢀ1, 3.8 and 3.2
mg mLꢀ1 and 3.2 and 3.0 mg mLꢀ1, respectively. Peptides 29–36
represent the replacement of R4 position with hydrophilic
amino acids. Analog 36 containing all D-amino acids showed the
highest potency against D6 and W2 strains with IC50 values of
1.9 and 1.7 mg mLꢀ1, respectively. While analogs 33 (R4 ¼ Lys)
showed good activity against D6 and W2 strains with IC50 values
of 2.0 and 1.7 mg mLꢀ1, respectively. In general, peptides have
displayed higher potency against drug-resistant W2 strain
(Table 2).
The activities of peptides 37–62 (series 2) are shown in
Table 3. In the case of peptides 37–43, in which N-terminus
Fig. 6 PLM II assay of selected peptides.
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Table 5 Analysis of 10 co-crystallized structures to identify critical amino acids necessary for the plasmepsin inhibitiona
Similarity with
˚
PDB ID Resolution (A) Ligand Ki/IC50 (nM) peptide ligand Asp34 Val78 Asn76 Leu131 Ser79 Ser218 Tyr192 Gly216 Phe111
2BJU
2IGX
1LF2
1LEE
1W6H
1XE5
2IGY
1ME6
1LF3
1XE6
1.56
1.70
1.80
1.90
2.24
2.40
2.60
2.70
2.70
2.80
IH4
A1T
R37
R36
TIT
5FE
A2T
IVS
34
54
30
18
0.08
0.08
0.21
0.21
0.20
0.13
0.09
0.12
0.15
0.17
pi
pi
pi
pi
H
H
H
H
H
H
H
H
H
H
H + pi
H
H
H
H
0.5–2.2
H
H
H
H
H
H
H
H
pi
pi
H
H
H
113
100
H
H
H
H
H
EH5
5FP
H
H
H
a
H, hydrogen bond donor or acceptor; pi, p–p interaction.
amino acid Lys is replaced, analog 37 (R1 ¼ Lys) produced Dab) and 60 (R4 ¼ Lys) showed activity against D6 and W2
activity against D6 and W2 strains and displayed IC50 values of strains with IC50 values of 2.6 and 1.8 mg mLꢀ1, respectively.
2.0 and 1.6 mg mLꢀ1, respectively. While analog 40 (R1 ¼ Arg) Analog 57 (R4 ¼ His) also exhibited modest activity against D6
showed good activity against D6 and W2 strains with IC50 values and W2 strains by producing IC50 values of 3.2 and 3.0 mg mLꢀ1
of 2.6 and 1.8 mg mLꢀ1, respectively. All other analogs of this respectively.
,
series were inactive against D6 and W2 strains. The peptide
The antiplasmodial activities of peptides of series 3 are
analog 43 containing D-Lys was inactive at the highest test presented in Table 4. The peptides 64 [R1 ¼ Phe(4-Cl)] and 65
concentration of 10 mg mLꢀ1. For peptides 44–49, in which [R1 ¼ Phe(4-NH2)] exhibited promising activity against D6 and
substitutions at the 2-position of lead peptide II were carried W2 strains with IC50 values of 2.0 and 1.6 mg mLꢀ1 and 2.6 and
out, analog 44 [R2 ¼ Phe(4-NH2)] and 46 [R2 ¼ Phe(4-Cl)] were 1.8 mg mLꢀ1, respectively. While analogs 63 (R1 ¼ Phe) and 66
the most potent peptides against D6 and W2 strains, and [R1 ¼ Phe(4-t-Bu)] showed activity against D6 and W2 strains
produced IC50 values of 1.6 and 1.4 mg mLꢀ1, respectively. It was with IC50 values of 4.0 mg mLꢀ1, respectively. For peptides 70–
observed that replacement of Phe with unnatural side-chain 75, in which substitutions at the position 2 of lead peptide III
containing residues Cha (45) and homoPhe (48) results in were carried out, analog 74 (R2 ¼ Dab) exhibited modest activity
total loss of activity. In the case of replacement of Phe at the against D6 and W2 strains with IC50 values of 3.2 and 3.0 mg
position 3 with Phe(4-t-Bu) (53) promising activity against D6 mLꢀ1, respectively. While analog 71 (R2 ¼ His) also showed
and W2 strains with IC50 values of 2.0 and 1.6 mg mLꢀ1
respectively was observed. The remaining substitution at this mg mLꢀ1, respectively.
,
activity against D6 and W2 strains with IC50 values of 4.7 and 4.2
position proves to be non-consequential for the activity. The
For peptides 76–81, in which Phe at 3 position is replaced,
peptides also produced selectivity index between >7.1 to 3.1 for peptide analog 78 [R3 ¼ Phe(4-t-Bu)] was the most promising
both tested strains. Replacement at C-terminus with other peptide and produced IC50 values of 1.6 and 1.4 mg mLꢀ1
,
hydrophilic amino acids, produced 56 (R4 ¼ Arg) exhibiting respectively against D6 and W2 strains (Table 4, series 3). While
encouraging activity against D6 and W2 strains with IC50 values analog 76 [R3 ¼ Phe(4-Cl)] showed encouraging activity against
of 2.0 and 1.6 mg mLꢀ1, respectively. While peptides 59 (R4 ¼ D6 and W2 strains with IC50 values of 2.0 and 1.6 mg mLꢀ1
,
respectively. Analog 77 [R3 ¼ Phe(4-NH2)] also showed modest
IC50 values of 2.6 and 1.8 mg mLꢀ1, respectively against D6 and
W2 strains. Substitutions at the C-terminus in lead peptide III
gave seven analogs 82–88. Analog 82 (R4 ¼ Arg) exhibiting
promising activity against D6 and W2 strains with IC50 values of
1.6 and 1.4 mg mLꢀ1, respectively. Analogs 84 (R4 ¼ Lys) and 85
(R4 ¼ Dab) showed modest activity against D6 and W2 strains
with IC50 values of 2.6 and 1.8 mg mLꢀ1, respectively. The
peptides belonging to this series, in agreement with the
observation made for most of the peptide analogs screened were
found more effective against drug-resistant W2 strain of P.
falciparum.
Cytotoxicity evaluation
All synthesized peptides were also evaluated for cytotoxicity in
Fig. 7 The docking pose of co-crystallized ligand into the active site of
plasmepsin II protein (PDB ID: 1W6H).
mammalian cell line to determine their safety prole. The in
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Table 6 The docking pose analysis of synthesized plasmepsin II inhibitorsa
Peptide
PSA
VAL78 SER79 THR114 TYR192 ASP34 GLU36 ASN76 ASP130 LEU131 ASP214 GLY216 THR217 SER218
63
64
65
66
76
77
78
71
74
84
85
57
37
53
46
44
59
60
202.66
202.66
228.68
202.66
202.66
228.68
202.66
205.31
202.66
202.66
202.66
205.32
202.66
202.66
202.66
228.68
202.66
202.66
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
Co-crystal 106
1
a
1, presence of hydrogen bond interaction.
vitro cytotoxicity of analogs was determined against VERO results are shown in the Fig. 6 below. The compounds were
noncancerous mammalian cells (obtained from ATCC, Amer- assayed at a concentration of 2 mM. The tests were done twice
ican Type Culture Collection) by neutral red assay.29 None of the and the % activity values given above are averages of the
peptides exhibited cytotoxicity at the tested concentration of duplicate determinations. The maximum inhibition was 25%
10 mg mLꢀ1
.
for peptide 78.
Enzyme inhibition assay against PLM II
Molecular modeling
Six compounds from the series were tested in enzymatic A large number of crystal structures of plasmepsin II are
assays against the catalytic activity of plasmepsin-II from P. reported in the protein data bank (PDB). From the available
falciparum. The compounds were rst dissolved in DMSO and eighteen crystal structures of plasmepsin II (PLM II), ten PLM
then diluted into buffered solutions at pH 4.5 for assay. The II and inhibitor complex crystal structures were analyzed to
Table 7 The IC50 value, docking score, glide score, glide emodel and glide energy for the synthesized plasmepsin II inhibitors
P. falciparum
P. falciparum
Peptide
D6 IC50 (mg mLꢀ1
)
W2 IC50 (mg mLꢀ1
)
Docking score
Glide score
Glide emodel
Glide energy
78
46
44
64
76
37
53
65
77
84
85
59
60
74
57
63
66
71
1.6
1.6
1.6
2.0
2.0
2.0
2.0
2.6
2.6
2.6
2.6
2.6
2.6
3.2
3.2
4.0
4.0
4.7
1.4
1.4
1.4
1.6
1.6
1.6
1.6
1.8
1.8
1.8
1.8
1.8
1.8
3.0
3.0
4.0
4.0
4.2
ꢀ9.65355
ꢀ10.4831
ꢀ10.1949
ꢀ8.44164
ꢀ11.0705
ꢀ9.08432
ꢀ10.0685
ꢀ9.29018
ꢀ9.28766
ꢀ9.25124
ꢀ8.70013
ꢀ9.69515
ꢀ9.58133
ꢀ9.12786
ꢀ9.91432
ꢀ9.31489
ꢀ8.80126
ꢀ9.17562
ꢀ9.93805
ꢀ10.6687
ꢀ10.3805
ꢀ8.72614
ꢀ11.355
ꢀ130.391
ꢀ138.429
ꢀ135.495
ꢀ112.604
ꢀ149.902
ꢀ119.042
ꢀ117.491
ꢀ127.521
ꢀ117.716
ꢀ116.79
ꢀ70.5439
ꢀ68.6636
ꢀ70.7052
ꢀ61.9412
ꢀ76.0211
ꢀ64.7812
ꢀ65.8653
ꢀ67.3476
ꢀ64.1044
ꢀ68.161
ꢀ9.86242
ꢀ10.2541
ꢀ9.57468
ꢀ9.57216
ꢀ9.53574
ꢀ8.98463
ꢀ9.88075
ꢀ9.76693
ꢀ9.43276
ꢀ11.063
ꢀ118.902
ꢀ136.38
ꢀ60.6688
ꢀ70.5735
ꢀ69.2143
ꢀ66.0703
ꢀ80.2347
ꢀ70.6532
ꢀ61.1545
ꢀ69.4552
ꢀ120.921
ꢀ120.271
ꢀ147.77
ꢀ9.89719
ꢀ9.08576
ꢀ9.86112
ꢀ126.601
ꢀ106.702
ꢀ132.68
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Ser218 are critical amino acids, and necessary for PLM inhibi-
tion. The docked conformation of the co-crystallized and one of
the most potent synthesized peptide 46 [K-Phe(4-Cl)-F-O] is
shown in Fig. 8.
The co-crystallized ligand has shown eight hydrogen bond
interactions (four HBA and four HBD) with the Val78, Ser79,
Tyr192, Ser218, Gly36, Asn76, Asp214 and Thr217, along with
one p–p interaction with the Phe111. The peptide 46 also
showed the docking score of ꢀ10.4831 and the emodel score of
ꢀ138.429. The peptides 44, 53 and 76 also produced high
docking scores of ꢀ10.3805, ꢀ10.2541 and ꢀ11.355; however
their emodel scores were low. Most of the synthesized peptide
showed hydrogen bond interaction with carboxylic groups of
Asp34 and Asp214. In case of the least potent inhibitor 71, the
hydrogen bond interactions with the critical residues Val78,
Tyr192, Asn76 and Thr217 were missing, possibly resulting in
low activity (Fig. 9).
Fig. 8 The docking pose of most potent synthesized inhibitor (46) into
the active site of plasmepsin II protein (PDB ID: 1W6H).
identify critical amino acids, necessary for the inhibition of
plasmepsin. Table 5 shows hydrogen bond and p–p inter-
action formed by the inhibitors at the active site of PLM-II.
The available crystal structures were also analyzed for their
resolution and presence of similar ligands to that of the
synthesized peptides. Aer a detailed analysis, PDB ID:
1W6H was selected for performing molecular docking anal-
ysis, as crystallized inhibitors were found to be more potent
(2.2 nM) and showing high Tanimoto similarity (0.20) with
the synthesized ligands.
Conclusions
In summary, a library of short peptide-based inhibitors was
designed by applying a rational and directed approach. To
synthesize peptides, we rst optimized the protocol for the
amino acids loading on to the Wang and 2-chlorotrityl chloride
resin. The most potent peptides of this work exhibit IC50 value
of 1.6 mg mLꢀ1 and 1.4 mg mLꢀ1 against chloroquine-sensitive
(D6) and chloroquine-resistant (W2) strain of P. falciparum.
Importantly, higher antimalarial activity against the drug-
resistant strain of P. falciparum was observed for the tested
peptides, indicating their potential in the treatment of drug-
resistant malaria parasite. The peptides appear to exhibit anti-
malarial activity by inhibiting PLM II. The lack of cytotoxicity in
all tested peptides provides the evidence of their safety prole
and makes these novel compounds promising for further
development as antimalarial agents.
The docking protocol was validated by re-docking of mini-
mized conformation of co-crystallized ligand. The RMSD value
between the docked conformation and the crystallized confor-
˚
mation was found to be 0.998 A, which suggests that the set
docking protocol is efficient to predict the bioactive conforma-
tion of the synthesized inhibitors (Fig. 7).
The Glide module failed to generate docking pose for some
of the inhibitors having a total polar surface area (TPSA) higher
˚
than 228 A (Table 6). The top ten poses were analyzed for each
inhibitor. The docked pose was selected, if the peptide showed a
similar pattern as that of the co-crystallized ligand. The active
site residues involved in binding of synthesized ligand are
shown in Table 6. Table 7 shows IC50 value, docking score, glide
score, glide emodel and glide energy for the selected eighteen
synthesized ligands. The crystal structure and molecular dock-
ing analysis reveal that the Val78, Ser79, Asp34, Asp214 and
Experimental section
Materials
Amino acids, coupling reagents, N,N-diisopropylethylamine
(DIEA), and triuoroacetic acid (TFA) were purchased from
either Chem-Impex International or Nova Biochem (Merck
Ltd.). All solvents used for synthesis were of analytical grade and
were used without further purication unless otherwise stated.
All other reagents were of analytical grade.
Peptide synthesis and purication
The peptides were synthesized using the Fmoc SPPS protocol
on a fully automated peptide synthesizer (Aapptec, Focus
XC). The coupling reactions were carried out on the 2-chlor-
otrityl chloride resin using Fmoc-protected amino acids (3
equiv.) and TBTU (3 equiv.) as a coupling reagent in the
presence of DIEA (5 equiv.) in DMF as solvent for 120 min. N-
terminus deblocking was carried out with 20% piperidine
(two times) in DMF for 5 min. The peptide was washed with
DMF (4 ꢁ 5 mL), and coupling and deprotection cycles were
Fig. 9 The docking pose of the least potent synthesized inhibitor (71)
into the active site of plasmepsin II protein (PDB ID: 1W6H).
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repeated until required sequence length was obtained. The Receptor Grid Generation tool. Extra precision (XP) Glide algo-
removal of peptide from the resin and deprotection of the rithm was validated to perform docking studies of the synthe-
side chain protective groups was achieved simultaneously by sized molecules, by re-docking the co-crystallized inhibitor into
the reaction with a cleavage cocktail combination of 5% the grid of the target. LigPrep was used for energy minimiza-
phenol, 2% triisopropylsilane (TIPS) and 5% H2O in TFA for tions of the synthesized ligands with the OPLS_2005 force eld.
2.5 h. Aer removal of the protecting groups and resin, The default settings for scaling the van der Waals radii were
peptides were precipitated by addition of cold diethyl ether selected: a scaling factor of 0.8 and a partial charge cut-off of
and ltered. The crude peptides were dissolved in a mixture 0.25 with no constraints were dened for docking of synthe-
of H2O–CH3CN (1 : 1, v/v) and puried by RP-HPLC on the sized ligands.
Shimadzu Prominence A-8 HPLC system, using a Phenom-
enex column (C-18, 250 ꢁ 21 mm, 10 mm), operating at 220
nm with a ow rate of 10 mL minꢀ1 to afford nal peptides in
90–100% purity, unless otherwise stated. The injected
sample concentration was 10 mg of peptide per mL in H2O–
CH3CN (1 : 1, v/v). The mobile phase A was 0.1% TFA in H2O,
and phase B was 0.1% TFA in CH3CN using a linear gradient
of 5 to 95% B in 40 min.
Abbreviations
AA
Amino acid
Abu
Ala
Arg
Cha
Aminobutyric acid
Alanine
Arginine
Cyclohexylalanine
CH3CN Acetonitrile
PLM II inhibitory assay
Dab
2,4-Diaminobutyric acid
The assay for catalytic activity was developed by Westling et al.30
DBU
1,8-Diazabicyclo[5.4.0]undec-7-ene
Dichloromethane
Briey, the gene for the proenzyme form of PLM II of P. falci- DCM
DCE
1,2-Dichloroethene
parum was cloned into pET3a and expressed in BL21 (DE3)
pLysS cells. Inclusion bodies were isolated and washed by
centrifugation through a 27% sucrose gradient. The inclusion
bodies were solubilized with 8 M urea and the protein refolded
by dialysis versus a buffer of 20 mM Tris at pH 8.0. The protein
was puried by anion exchange chromatography and stored at
DMAP 4-Dimethylaminopyridine
DMF Dimethylformamide
DMSO Dimethyl sulfoxide
Fmoc 9-Fluorenylmethoxycarbonyl
His
Histidine
HPLC High-performance liquid chromatography
IC50
ꢂ
4 C.
Inhibitory concentration that affords 50% inhibition
The proenzyme was added to the assay buffer (sodium
acetate, pH 4.5) for three-minute incubation, during which the
proenzyme smoothly converts to the mature and catalytically
active enzyme. The enzyme does not digest itself, so the
concentration of the proenzyme added can be taken as the
concentration of the enzyme produced. Aliquots of compounds
to be tested were added to the enzyme for a ve-minute pre-
incubation to permit complex formation to proceed and then
the sample was added to the assay substrate and mixed to
initiate cleavage. Conversion of substrate to products was
monitored by the decrease in absorbance at 300 nm in an
UV/Vis spectrophotometer as a function of time. All compounds
were added so that a nal concentration of 2% DMSO was
present and this amount was also used in the control reaction to
establish full activity. It was previously determined that 2%
DMSO has negligible effect on the catalytic activity.
of microbial growth
Isoleucine
Lysine
Minimum inhibitory concentration
1-Napthylalanine
Norleucine
N-Methyl-2-pyrrolidone
Ornithine
Ile
Lys
MIC
Nal
Nle
NMP
Orn
Phe
Ser
Phenylalanine
Serine
tert-Butyl
t-Bu
TEA
Triethylamine
TBTU O-(Benzotriazol-1-yl)-N,N,N0,N0-
tetramethyluroniumtetrauoro-borate
THF
TFA
Thr
TIPS
Trp
Tyr
Tetrahydrofuran
Triuoroacetic acid
Threonine
Triisopropylsilane
Tryptophan
Molecular modeling
1W6H was selected to carry out molecular docking analysis on
the basis of the resolution and structural similarity with the
synthesized inhibitors. The Protein Preparation Wizard and
LigPrep modules support in the generation of input les, which
are essential to carry out the molecular docking study. During
protein preparation hydrogen atoms were added to the target,
water molecules removed, the protonation states for histidine
Tyrosine
Valine
Val
Acknowledgements
residues were optimized and the hydrogen atoms were mini- Dr Amit Mahindra and Rahul P. Gangwal are thankful to the
˚
˚
mized up to 0.30 A RMSD. The grid of 10 A was generated Council of Scientic and Industrial Research (CSIR), New Delhi
around the co-crystallized ligand at the active site by the for the award of a Senior Research Fellowship.
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