De novo lead optimization of triazine derivatives identifies potent antimalarials

Article abstract of DOI:10.1016/j.jmgm.2016.10.022

Malaria is a life-threatening disease caused by Plasmodium parasites among which Plasmodium falciparum is the most deadly. Due to the widespread resistance of the current antimalarial drugs, intense research efforts are focused on identification of new an

Full text of DOI:10.1016/j.jmgm.2016.10.022

Journal of Molecular Graphics and Modelling 71 (2017) 96–103
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Journal of Molecular Graphics and Modelling
journal homepage: www.elsevier.com/locate/JMGM
Topical Perspectives
De novo lead optimization of triazine derivatives identifies potent
antimalarials
Ashutosh Shandilyaa, Nasimul Hodab, Sameena Khanc, Ehtesham Jameelb,
Jitendra Kumarb, B. Jayarama,d,∗
a Department of Chemistry and Supercomputing Facility for Bioinformatics & Computational Biology, Indian Institute of Technology, Delhi Hauz Khas, New
Delhi, 110016, India
b B.R. Ambedkar Bihar University, Muzaffarpur, India
c Translational Health Science and Technology Institute, Gurgaon, India
d Kusuma School of Biological Sciences, IIT Delhi, New Delhi, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 31 August 2016
Received in revised form 11 October 2016
Accepted 14 October 2016
Available online 13 November 2016
Malaria is a life-threatening disease caused by Plasmodium parasites among which Plasmodium falciparum
is the most deadly. Due to the widespread resistance of the current antimalarial drugs, intense research
efforts are focused on identification of new and potent antimalarials. We report here, a structure based
drug discovery strategy for design of a series of effective and novel triazine based antimalarials. The X-ray
structure of P. falciparum methyl transferase (PfPMT) is used as a target as it is unique to the parasite. The
triazine molecules designed and synthesized showed low micro-molar activity against malarial parasite
cell lines. Molecular dynamics simulations on the PfPMT-inhibitor complex shed light on the inhibition
mechanism for further optimization of the lead compounds.
Keywords:
Malaria
Triazine derivatives
PfPMT
© 2016 Elsevier Inc. All rights reserved.
Molecular dynamics simulations
1. Introduction
phosphocholine (pCho) [8]. Biochemical studies uncovered that
PfPMT action was repressed by S-adenosyl homocysteine (SAH) and
Plasmodium falciparum is the most lethal form accounting for
a majority of mortality resulting from malaria [1]. Treatment of
malaria has changed in the course of recent decades due to a decline
in medication efficacy and a resurgence of malaria in tropical
regions [2]. Rapid rise of resistance to the most potent quinolones,
antifolates [3] (such as sulphadoxine/pyrimethamines) and recent
reports of potential artemisinin resistant parasites [4] have spurred
an urgent need for the development of new and effective anti-
malarial molecules that target important parasite functions and
have the potential to act against multi-drug-resistant plasmodium
strains to prevent malaria pathogenesis and transmission.
Recent studies on P. falciparum have unraveled new metabolic
pathways for the synthesis of the parasite phospholipids and fatty
acids [5–7]. Plasmodium falciparum phosphoethanolamine methyl
transferase (PfPMT) is an adenosyl methamine (SAM) dependent
methyl transferase that catalyzes phosphoetheanolamine (pEa) to
by phosphocholine [9]. Genetic studies in P. falciparum showed that
PfPMT assumes a critical part amid the intraerythrocytic life cycle of
the parasite [10]. Knock out of the PfPMT totally annuls the biosyn-
thesis of phosphatidylcholine by means of (serine-decarboxylase-
phosphoethanolamine-methyltransferase) SDPM pathway [11].
Growth and propagation of parasite requires large amount of
phospholipids, which are synthesized from plasma fatty acids.
Phosphocholine (pCho) and phosphoethanolamine (pEa) make up
for around 80% of phospholipids in Plasmodium falciparum [12].
Lipid metabolism has been reported to be dramatically increased
during the development of the malaria parasite, contributing
mainly to membrane biogenesis which is almost nonfunctional
in the uninfected red blood cells [13,14]. This indicates that the
metabolic pathways for the synthesis of major Plasmodium fal-
ciparum phospholipids can be excellent targets as supported by
several studies [15]. Plasmodium uses a plant like phosphobase
pathway for the biosynthesis of pCho as a metabolic precursor
for phospholipid synthesis [15]. The overall reaction catalyzed by
the PfPMT resembles that of other N-methyltransferases; however,
mechanism of transfer reaction differs [7–9,15,16]. Importantly,
this phosphobase methylation pathway and hence the PfPMT which
∗
Corresponding author at: Department of Chemistry and Supercomputing Facility
for Bioinformatics & Computational Biology, Indian Institute of Technology, Delhi
Hauz Khas, New Delhi, 110016, India.
E-mail address: bjayaram@chemistry.iitd.ac.in (B. Jayaram).
http://dx.doi.org/10.1016/j.jmgm.2016.10.022
1093-3263/© 2016 Elsevier Inc. All rights reserved.

A. Shandilya et al. / Journal of Molecular Graphics and Modelling 71 (2017) 96–103
97
is essential for the normal growth and survival of the Plasmodium
is not found in humans making it a viable target [8,10,17].
NPT conditions for 100 ns. A 2 femto second time step was used
for integrating the equations of motion. Periodic boundary con-
ditions were applied throughout the MD simulations, along with
PME summation [34] for treating electrostatics. The temperature
was kept constant by coupling heat bath through the Berendsen
algorithm [35] using separate solute and solvent scaling. Pressure
was adjusted by isotropic position scaling using a Berendsen like
algorithm. Covalent bonds to hydrogen atoms were constrained
by the SHAKE algorithm [36]. Convergence of energy and density
were monitored. A similar method has been applied to explain
allosteric changes in domains of PfATP6 enzyme in presence of
artemisinin [37] and specific inhibition of glycosidases [38]. The
initial 10 ns was treated as equilibration and the subsequent 225 ns
was treated as production phase for structural and energy analy-
ses. Overall stability was analyzed by RMSD plot (Supplementary
Fig. S1) and fluctuating residues were identified using RMSF plot
(Supplementary Fig. S2).
Although, potential chemical diversity space is estimated to be
around 1060 molecules [18], at present, only a limited number
of chemical scaffolds and targets are in clinical use for treating
malaria. Our idea is to develop lead molecules by identifying dis-
crete components showing molecular recognition to PfPMT. We
started with low molecular weight chemical scaffolds on the basis
of their ability to bind to the target protein. The diverse biolog-
ical activities observed for different molecules containing 1, 3, 5
Triazine unit prompted us to discover their new potential as anti-
malarials. 4-aminoquinoline-triazine based hybrids have shown
activity on a range of cell lines and apoptosis as the mode of growth
inhibition [19]. Some triazine derivatives acted as potent (phos-
phoinositide 3-kinase inhibitor) PI3K inhibitors and were used for
treatment of different types of cancer [20,21]. Lamotrigine (a tri-
azine derivative) is an FDA approved antiepileptic drug [22]. Here,
we describe the identification of triazine derivatives that show low
micromolar anti-malarial activities against P. falciparum parasite.
3. General procedure for the synthesis of triazine [39]
2. Materials and methods
3.1. Analogues P
2.1. System preparation
The stirred solution of 5.3 g (29.0 mmol) of cynuric chloride in
200 mL of DCM was cooled to −15 ◦C. A solution of 1.0 eq. of appro-
priate amine and 1.0 eq. of N,N-diisopropylethylamine in 40 mL of
DCM was added drop-wise to the cynuric chloride solution. After
the completion of the addition, the reaction mixture was stirred
at 0 ◦C for 0–4 h. The reaction mixture was quenched by pouring
it into 150 mL of a 5% aq. citric acid solution and extracted with
DCM (2 × 100 mL). The organic layers were combined, dried over
Na2SO4 and concentrated in vacuo. Purification of the compound P
was done by silica gel column chromatography.
Crystal structure of the P<!–Pf should be italics–!>fPMT with
sinufungin [PDB: 3UJ8] was obtained from the Protein Data Bank
[23]. The crystal structure contained several missing atoms. The
missing residues were modelled and H- atoms were added by
“Leap” module of AMBER suite for correct ionization and tautomeric
states of amino acid residues. Structure so obtained was used for
all the docking and simulation studies. The ligand molecule struc-
tures were built by Marvin sketch [24]. Partial charges of ligand
for electrostatic calculations were deduced by AM1 methods [25].
The structures of these compounds are given in Table 1. The geom-
etry of the ligand molecule is optimized and the target enzyme
energy minimized via alternate steepest descent and conjugate gra-
dient methods using AMBER 14, a comprehensive suite for carrying
molecular dynamics simulations.
3.2. Analogues q
The stirred 0.1 M solution of monosubstituted dichlorotri-
azine intermediate P in either DMF or THF was cooled to 0 ◦C
followed by drop-wise addition of a solution of 1.2 eq. of N,N-
diisopropylethylamine and 1.1 eq. of the appropriate amine in THF.
The reacting mixture was stirred at 0 ◦C for 10 min after the addition
was completed and then allowed to warm at 25 ◦C for 4–12 h. The
reaction mixture was poured into water and extracted with EtOAc
(2 × 100 mL) and finally with brine. The organic layers were com-
bined, dried with Na2SO4 and concentrated in vacuo. The obtained
crude residue Q was purified by silica gel chromatography.
2.2. Docking
Docking of ligands to the enzyme were carried out using Par-
dock module of Sanjeevini, a comprehensive drug design suite
[26–30]. Docking takes place in four steps. The protocol identi-
fies best possible grid in a radius of 3 Å around the geometric
center of the active site/reference points. Energy of each atom of
ligand is pre-calculated around the generated protein grid. Con-
figurational sampling of the ligand molecule inside the active site
is done via Monte Carlo algorithm. Best docked structure is iden-
tified by energy criterion. The docking is followed by empirical
estimates of binding free energies of the top 8 docked structures
using Bappl scoring function [30]. This docking method is freely
accessible at http://www.scfbio-iitd.res.in/dock/pardock.jsp. These
docked structures were taken as input for molecular dynamics sim-
ulations.
3.3. Analogues r
To stirred 0.1 M solution of disubstituted monochlorotriazine
Q in anhyd. DMF or either 1:1 DMF/THF or DMF/CH3CN at room
temperature, a 1.3 eq. of nucleophile and 1.5 eq. of appropriate base
was added. The reacting mixture was stirred to reflux for 8–12 h and
then cooled to room temperature. The reaction mixture was poured
into 200 mL of EtOAc or 1:1 mixture of EtOAc/Ether and extracted
with brine (2 × 75 mL). The organic layers were combined, dried
(Na2SO4), and concentrated in vacuo. The final crude compound R
was purified by silica gel column chromatography.
2.3. Molecular dynamics
All MD simulations were carried out using AMBER 14 [31] using
FF99SB [32] forcefield. The complex obtained from Pardock was
solvated in a box of TIP3P [33] water molecules and minimized for
an additional 2500 steps (1000SD + 1500CG). The size of the system
is ∼75,000 atoms. MD simulations were started by slowly heating
the solvent to 300 K over a period of 500 ps keeping the com-
plex fixed. After this, the entire system was gradually brought to
300 K in another 500 ps. The simulation was then carried out under
3.4. Parasite growth inhibition assay
To assess the effect of inhibitors on malaria parasite growth in
vitro, synchronous ring-stage 3D7 strain P. falciparum was cultured
in a 96 well plate. Compounds were dissolved in DMSO and were
further diluted in culture medium to obtain the required concen-
trations. Compounds were tested in varying concentrations ranging

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Table 1
List of synthesized molecules.
Molecule
Ar
R1
R2
1
2
3
4
5
6
7
8
9
10
11
12
13
14
from 0 to 100 ␮M. P. falciparum proliferation in human erythro-
cytes was measured utilizing SYBR green I dye DNA staining assay
as reported earlier [40]. After 48 h of growth, parasites were lysed
using lysis buffer containing SYBR Green I by adding 100 ␮L of lysis
buffer to each well and contents were mixed. After 1 h of incuba-
tion in dark at room temperature, fluorescence was measured using
multiwell plate reader (Victor3, Perkin Elmer) with excitation and
emission wavelength bands centered at 485 and 530 nm respec-
tively. IC50 values were obtained by plotting fluorescence values
against the inhibitor concentrations. Chloroquine was used as a
positive control in all experiments and each inhibitor was tested
in triplicates.
ing 10% fetal bovine serum, 02% sodium bicarbonate and 50 mg/ml
gentamycin. Briefly, cells (104cells/200 mL/well) were seeded into
96 well plate (tissue culture grade, flat bottom) in complete cul-
ture medium. Inhibitors were added after overnight seeding and
incubated for 24 h at 37 ◦C in humidified atmosphere with 5%
CO2. DMSO (final concentration 10%) was used as positive control.
20 ␮L of MTT stock (5 mg/ml in 1× phosphate-buffered saline) was
added to each well and incubated for 4 h. Supernatant was removed
after spinning the plate at 1500 rpm for 5 min and 100 ␮L of the
stop agent DMSO was added. Formation of formazan (chromogenic
product from MTT reduction) indicated survival and was measured
at 570 nm. The dose response curves were analyzed for toxicity.
3.5. Cytotoxicity activity measurement
4. Results and discussion
Mammalian cell line (HeLa) was used to determine the toxicity
of selected top inhibitors from parasite growth inhibition assay by
using MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium
Bromide] assay. Cells were cultured in complete RPMI contain-
The X-ray crystal structure (1.55 Å resolution) of PfPMT in com-
plex with inhibitor AdoMet (sinefungin) was employed in this study
for inhibitor design [8]. The PfPMT monomer consists of an alpha
(␣)-helical lid domain and a canonical binding domain defined by a

A. Shandilya et al. / Journal of Molecular Graphics and Modelling 71 (2017) 96–103
99
central beta (␤) sheet surrounded by two ␣-helical regions. Broadly
PfPMT fold shares high similarity with N-methyltransferases from
Mycobacterium [41] and have a conserved AdoMet binding domain.
Active site of PfPMT is identified by the position of sinefungin as
it bifurcates the alpha helical domain and the central beta sheet of
the enzyme. Active site interior consists of three aspartic acids (Asp
−61, 85, and 110) and one basic residue Arg 179. His 132 and Tyr 19
are situated on the either side of AdoMet. In the PfPMT- sinefungin
complex, the position of sinfungin at the interface of two donors
clearly describes the active site which comprises LEU 14, TYR 19,
SER 37, ASP 85, GLY 65 and ASP 128, HIS 132. Within the PfPMT
active site TYR 19 and HIS 132 are positioned as a catalytic dyad
[8].
(Table 2) also indicate molecule 10 to be a good inhibitor. We inves-
tigated the plausible reason for this specific binding and inhibition
via molecular dynamics (MD) simulations. The aim of the MD sim-
ulations was to observe structural and dynamic changes in the
enzyme in the presence of the ligand and to obtain a more precise
description of the binding modes of ligand to the enzyme in the
presence of solvent considering the flexibilities of both receptor
and ligand [37,38].
An analysis of the MD trajectories shows that residues of N
terminal of the PfPMT enzyme which encompass helical and loop
regions move towards the ligand in such a way as to affect a clo-
sure of the active site cleft, stabilizing the ligand molecule inside
the cavity (Supplementary Fig. S4). Methylation of phosphobase is
assisted by conversion of SAM to SAH which requires the presence
of key residues ASP 118, HIS 132 and TYR 19 as mentioned ear-
lier. A closer look at the active site residues (Fig. 2a) indicates that,
HIS 132 and TYR 19 are in the close proximity (2.2 Å) in the crystal
structure (3UJ8). However, during simulation, molecule 10 moves
laterally so as to come in between these two residues and make
them part away (7.3 Å). Phosphobase also drifted from the active
site along with the position of a critically important residue ASP
118 (Fig. 2b).
To examine in detail the ligand-receptor interactions of PfPMT-
triazine derivative complex and to estimate the dynamic stabilities
of the hydrogen bonds facilitating the binding of inhibitor in the
active site of PfPMT, we calculated the time evolutions of the asso-
ciated interatomic distances which show that once HIS 132 and
TYR 19 are parted, they remain so for the entire length of simu-
lation (Fig. 3). Investigation of ligand binding region demonstrates
that molecule 10 forms directional hydrogen bonds with side chain
atoms of SER 37 and TYR 171 (TYR 171 not shown in image for
clarity) and main chain oxygen of ILE 76 throughout the entire tra-
jectory giving the ligand molecule stability in the complex (Fig. 4).
However hydrogen bonds with main chain oxygens of TYR 9 and
ASP 75 vanished indicating that these hydrogen bonding inter-
actions were weak (Fig. 5). Structural rearrangements in these
residues induced some new hydrogen bonds with main chain oxy-
gen of residue LEU131 and ring nitrogen of HIS 132. van der Waals
interaction with N termini residues from residue 4 to 10 also play
a crucial role in binding as these residues allow the helical region
to converge towards ligand which assist in binding of the ligand.
Favorable interactions from SER 37 and ILE 130 and LEU 131 also
stabilize the binding of the ligand (Supplementary Fig. S5).
Rapid screening of available databases against PfPMT resulted
in multi substituted ligands with one or two aromatic ring struc-
tures similar to pyridine and cynuric chloride. However, structure
of sinefungin is quite different from the resultant structures. We
compared the binding energies using docking and found that struc-
tures obtained from screening showed more interactions with the
active site residues as compared to sinefungin. Substitutions at
multiple places were relatively easier and would take only a few
steps of synthesis, with readily available cynuric chloride. Hence,
we initiated our study with a small fragment of triazine, as highly
complex triazine derivatives can be generated from simple reagents
under economically favorable reaction conditions from the starting
material. The substitution was governed by the complementarity
of the active site residues and the ligand molecule. Trifluorobenzyl
and cyclopropyl group and aniline or anisole group were substi-
tuted at two carbon atoms of the triazine ring. We designed a
number of molecules with diverse scaffolds for docking and scoring
on to the target enzyme in an attempt to identify new antimalar-
ial molecules with enhanced binding and Lipinski rule compliance.
The molecules designed against PfPMT were docked in the bind-
ing pocket defined by sinefungin in the crystal structure. Active
site of PfPMT is relatively spacious hence presence of bulkier group
with polar substituents should fit well in the active site. About 14
molecules were identified as good binders with binding energies
ranging from −8 to −12 kcal (Tables 1 & 2). HIS 132 and TYR 19 are
positioned at the opposite sides of the ligand which form a catalytic
latch that locks ligand in the active site and orders the site of cataly-
sis. HIS 132 functions as a general base to abstract a proton from the
hydroxyl group of TYR 19. The negatively charged oxygen of TYR 19
interacts with hydrogen on the substrate amine to distort the elec-
tronic configuration and orients the lone pair electrons towards
the methyl group of AdoMet to facilitate methylation of phospho-
base [9]. (Reaction mechanism is shown in supplementary Fig. S3.)
Residues within 4 Å of triazine derivatives were analyzed. TYR 19 is
involved in ligand binding due to ␲-␲ interaction with the ligand
while HIS 132 is involved in cationic − ␲ interaction with the aro-
matic ring of the ligand. Further QM/MM calculations suggested an
alternative mechanism involving ASP 128 which is critical for spe-
cific methylation of phosphoethanolamine (pEa) [16]. There are 9
residues near the ligand, including three hydrophobic residues (LEU
14, ILE 26, GLY 65 and LEU 131), two negatively charged residues
(ASP 85 and ASP 128), and three neutral residues (TYR 19, SER 37
and HIS132). Binding poses suggest interaction with neighboring
residues in the active site of PfPMT (Supplementary Fig. S6). As evi-
dent from Table 2, docking pose of molecule 10 showed the best
binding forming hydrogen bonds with side chains of TYR 19, SER
37, ASP 85 and ASP 128 (Fig. 1a & b). The results indicate that the
binding of triazine derivative to PfPMT is favored by a combina-
tion of polar and non-polar interactions. All the fourteen molecules
were further synthesized and checked for anti-plasmodium activity
in the in vitro parasite growth inhibition assay.
1 Synthesis
The compounds designed were synthesized via sequential
nucleophilic aromatic substitution reactions depicted in scheme
(Fig. 6) using known procedures with minor modifications. Addi-
tion of 1 equivalent of aromatic amines to cynuric chloride in
dichloromethane at −10 to 0 ◦C in the presence of proton scav-
enger afforded mono substituted triazine P, where one chlorine
is replaced by an aromatic amine. Subsequent reaction of P with
an appropriate nucleophile in the presence of 1 equivalent of base
resulted in the formation of disubstituted triazine Q and trisubsti-
tuted triazine R. The final displacement was introduced by reaction
with 2 equivalents of nucleophile and a base in refluxing dioxane.
Reaction scheme is shown in Fig. 6. Structure of triazine derivative
is confirmed by C13 and H1 NMR (Plots in Supplementary informa-
tion).
•
Parasite inhibition assay using the newly synthesized triazine
derivatives
According to the docking results (Table 2), molecule 10 showed
the best binding energy towards PfPMT. Experimental assays
All the synthesized molecules were checked for anti-
plasmodium activity in the in vitro parasite growth inhibition assay.

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A. Shandilya et al. / Journal of Molecular Graphics and Modelling 71 (2017) 96–103
Table 2
Computational predicted binding affinities and experimentally observed IC50 values of designed molecules.
Molecule
Binding Energies (kcal/mol)
(IC50 ␮M)
Molecule
Binding Energies (kcal/mol)
(IC50 ␮M)
Molecule 1
Molecule 2
Molecule 3
Molecule 4
Molecule 5
Molecule 6
Molecule 7
−9.2
∼21
∼2.4
∼17
∼1.5
∼71
∼10
∼77
Molecule 8
Molecule 9
Molecule 10
Molecule 11
Molecule 12
Molecule 13
Molecule 14
−9.6
∼58
∼28
∼0.8
>100
∼5
−10.1
−10.1
−10.8
−8.1
−10.1
−12.1
−8.5
−11.3
−9.8
−10.1
−9.9
>58
−9.1
∼54
Fig. 1. a. N-Methyl Transferase docked with a triazine derivative (Molecule 10) b. Interactions of Molecule 10 with neighboring residues.
Fig. 2. Snapshot of crystal structure bound with AdoMet (ligand) and phosphobase (left panel). Residues within 4 Å of AdoMet are shown. HIS 132 and TYR 19 are on either
side of ligand and in close proximity (2.2 Å). A snapshot of active site residues surrounding Molecule 10 showing HIS 132 and TYR 19 are parted away after simulation (right
panel).
Fig. 3. Interatomic distance variation carbon atom of Phenyl ring of HIS 122 and ring nitrogen atom of TYR 9 during the entire course of simulation.

A. Shandilya et al. / Journal of Molecular Graphics and Modelling 71 (2017) 96–103
101
Fig. 4. Interatomic distances of sidechain atom of ILE 76, SER 27 and TYR 171 with ligand throughout the 225 ns simulation showing these residues are coming closer to
ligand during the course of simulation.
Fig. 5. Interatomic distances of TYR 9 and ASP 75 with ligand shows distances are increasing with the ligand from the initial structure.
Fig. 6. Reagents: (i). ArNH2, DIPEA, DCM, −10–0 ◦C, 1–4 h, (ii) R1X, Triethylamine THF/DMF, 0 ◦C to room temp, 4–12 h, (iii) R2X, K2CO3, 120 ◦C, 12–24 h.
A good correspondence between computationally predicted bind-
ing energies and experimental IC50 values was observed (Fig. 7).
Eight of the tested inhibitors were found to have IC50 values lower
than 30 ␮M and five of the inhibitors exhibited IC50 values lower
than 10 ␮M (Fig. 8a and Table 2). We further tested whether the
inhibitors assayed above showed any cytotoxicity to mammalian
cells. We evaluated the cytotoxic effect using MTT assay against
HeLa cells (Fig. 8b). We observed that the two top inhibitors in
our parasite growth inhibition assays showed more than 10 fold
selectivity (molecule 10 ∼34 fold and molecule 4 ∼17 fold) for the
parasite cells over the human cell line. This high selectivity is con-
sistent with the fact that PfPMT homologue is absent in humans.
Parasite inhibition data together with the cytotoxicity data thus
provide us with lead compounds with potentially have high thera-
peutic value.
Fig. 7. Correlation plot between computational binding energies and log value of
experimental IC50 value.

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A. Shandilya et al. / Journal of Molecular Graphics and Modelling 71 (2017) 96–103
Fig. 8. Parasite growth inhibition assay. (a) Selected inhibitors from the docking analysis tested in the parasite growth inhibition assay using double dilution till 8 points
(100 ␮M–0.8 ␮M). (b) Cytotoxicity measurement for inhibitors.
4. Conclusion
Acknowledgement
A few newly designed molecules with triazine scaffold based on
docking studies with PfPMT were synthesized and assayed against
P.falciparum and were found to be good inhibitors with activi-
ties in the low micromolar to sub micromolar range. Molecular
dynamics simulations suggested that the inhibitor moves laterally
in the active site so as to come in between HIS 132 and TYR 19
physically blocking the substrate to be methylated, thus plausibly
inhibiting the enzyme. Our in silico analysis is further supported
by in vitro parasite growth inhibition assays. These molecules
show high specificity towards parasite cells as compared to human
cells. Detailed atomic interactions for compound molecule 10 (IC50
∼800 nM) observed therefore can serve as a platform for designing
next generation PfPMT inhibitors since this target enzyme is unique
to the malarial parasite.
We acknowledge the support from the Department of Biotech-
nology (DBT), Govt. of India, to the Supercomputing Facility for
Bioinformatics & Computational Biology, IIT Delhi.
Appendix A. Supplementary data
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.jmgm.2016.10.
022.
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