Benzothiophene carboxamide derivatives as inhibitors of Plasmodium falciparum enoyl-ACP reductase

Article abstract of DOI:10.1002/iub.553

Benzothiophene derivatives like benzothiophene sulphonamides, biphenyls, or carboxyls have been synthesized and have found wide pharmacological usage. Here we report, bromo-benzothiophene carboxamide derivatives as potent, slow tight binding inhibitors of Plasmodium enoyl-acyl carrier protein (ACP) reductase (PfENR). 3-Bromo-N-(4-fluorobenzyl)-benzo[b]thiophene-2-carboxamide (compound 6) is the most potent inhibitor with an IC50 of 115 nM for purified PfENR. The inhibition constant (K_i) of compound 6 was 18 nM with respect to the cofactor and 91 nM with respect to crotonoyl-CoA. These inhibitors showed competitive kinetics with cofactor and uncompetitive kinetics with the substrate. Thus, these compounds hold promise for the development of potent antimalarials.

Full text of DOI:10.1002/iub.553

IUBMB Life, 63(12): 1101–1110, December 2011
Research Communication
Benzothiophene Carboxamide Derivatives as Inhibitors of
Plasmodium falciparum Enoyl-ACP Reductase
1
2
1
1
*
Tanushree Banerjee , Shailendra Kumar Sharma , Neha Kapoor *, Vishnu Dwivedi ,
Namita Surolia3 and Avadhesha Surolia1
1National Institute of Immunology, New Delhi, India
2Molecular Biophysics Unit, Indian Institute of Science, Bangalore, India
3Jawaharlal Nehru Centre for Advanced Scientific Research, Jakkur, Bangalore, India
INTRODUCTION
Summary
Benzothiophene derivatives like benzothiophene sulphona-
Socioeconomic burden of malaria is increasing due to the
emergence of resistance towards the mainstay antimalarials
(1–4). There are variations in the frequency of resistance devel-
opment towards the existing drugs, which depends on the
genetic traits of both the host and the pathogen. Environmental
factors also play a deterministic role in the development of re-
sistance (5). Approximately 40% of world’s population is under
threat of malaria, which includes the countries in Asia, Africa,
Latin America, and South Pacific (6). Its causative agent is the
apicomplexan protozoan Plasmodium. The genus Plasmodium
has four species: malariae, vivax, falciparum, and ovale, which
infect humans. Recently, the monkey malaria parasite Plasmo-
mides, biphenyls, or carboxyls have been synthesized and have
found wide pharmacological usage. Here we report, bromo-ben-
zothiophene carboxamide derivatives as potent, slow tight bind-
ing inhibitors of Plasmodium enoyl-acyl carrier protein (ACP)
reductase (PfENR). 3-Bromo-N-(4-fluorobenzyl)-benzo[b]thio-
phene-2-carboxamide (compound 6) is the most potent inhibitor
with an IC50 of 115 nM for purified PfENR. The inhibition con-
stant (Ki) of compound 6 was 18 nM with respect to the cofac-
tor and 91 nM with respect to crotonoyl-CoA. These inhibitors
showed competitive kinetics with cofactor and uncompetitive
kinetics with the substrate. Thus, these compounds hold prom-
ise for the development of potent antimalarials. Ó 2011 IUBMB
IUBMB Life, 63(12): 1101–1110, 2011 dium knowlesi was also shown to infect humans. Among the
human malaria parasites, Plasmodium falciparum causes the
most fatal form of malaria viz. cerebral malaria. In the absence
of a vaccine and complete control on transmission vector, it is
imperative to develop new antimalarials.
Keywords bromo-benzothiophene; benzothiophene carboxamide;
antimalarials; Plasmodium.
Abbreviations FAS, fatty acid synthase; GPI, glycosylphosphatidyli-
nositol; PfENR, Plasmodium falciparum enoyl-ACP
reductase; NADH, nicotinamide adenine dinuleotide
Current insights on the Plasmodium’s metabolome have
uncovered different targets for the development of novel antima-
reduced; ADH, alcohol dehydrogenase.ACP: Acyl larials. Also, fatty acids biosynthesis enzymes are one of the im-
Carrier Protein DCC- Dicyclohexyl Carbodiimide
portant targets. Tremendous proliferative potential of Plasmo-
dium makes it essentially depend on abundant supply of fatty
acids. Fatty acids are required for membrane synthesis, lipid bio-
HoBt-1-Hydroxybenzotriazole DMF-Dimethy form-
amide ES-MS-Electron Spray Mass Spectrometry
DMSO: DimethylSulphonyl Oxide SDS/PAGE-So-
dium Dodecyl Sulphate/ Polyacrylamide Gel
Electrophoresis PDB: Protein Database
genesis and glycosylphosphatidylinositol (GPI) anchors of its
transmembrane proteins. In the case of Plasmodium, de novo syn-
thesis of fatty acids occurs by type II fatty acid synthase (FAS),
which is fundamentally different from type I FAS system of
humans (7–11). Using inhibitors (12) and type II FAS enzyme
knock-out parasite lines (13), it has recently been shown to be in-
dispensable for liver-stage parasite development which further
strengthens its significance in the parasite biology.
Additional Supporting Information may be found in the online ver-
sion of this article.
*These authors contributed equally to this work.
Address correspondence to: Avadhesha Surolia, National Institute of
Immunology, Aruna Asaf Ali Marg, New Delhi 11067, India. E-mail:
surolia@nii.res.in
In brief, the fatty acid synthesis starts with the formation of
malonyl-acyl carrier protein (ACP) by malonyl-CoA ACP trans-
acylase (FabD). Malonyl-ACP undergoes decarboxylative con-
Received 22 June 2011; accepted 7 July 2011
ISSN 1521-6543 print/ISSN 1521-6551 online
DOI: 10.1002/iub.553

1102
BANERJEE ET AL.
densation with acetyl-CoA by the enzyme b-ketoacyl-ACP syn- was done using Perkin Elmer API 165 Sciex mass spectrometer.
thase III (FabH) or acyl-malonyl-ACP condensing enzyme 1H and 13C NMR spectral analyses were performed on Bruker
(FabB or Fab F), respectively. This reaction is followed by nic- DRX 400 spectrometer with tetra-methylsilane as the internal
otinamide adenine dinucleotide phosphate reduced (NADPH) standard (d ppm). The following abbreviations were used to
dependent reduction, which is catalyzed by b-ketoacyl-ACP re- explain the multiplicities: s, singlet; d, doublet; t, triplet; dd,
ductase (FabG) forming b-hydroxy butyryl-ACP. b-Hydroxy bu- double doublet; m, multiplet, br, broad. Solvents and reagents
tyryl-ACP is then dehydrated to trans-2-butyryl-ACP (crotonoyl- were purified according to standard laboratory techniques.
ACP) by b-hydroxy acyl-ACP dehydratases (Fab A or Fab Z).
The last step is the nicotinamide adenine dinuleotide reduced General Procedure for Synthesis of Compounds 4–9. To a
(NADH) dependent reduction, of the enoyl-ACP catalyzed by cooled mixture (ice bath) of 3-bromobenzo[b]thiophene-2-car-
enoyl-ACP reductase (FabI) forming butyryl-ACP. Repetition of boxylic acid (2) (0.60 g, 2.33 mmol), corresponding arylalkyl
the elongation cycle 6–7 times yields a long acyl chain covalently amine (3.12 mmol), dicyclohexyl carbodiimide (DCC) (0.48 g,
linked to ACP. Plasmodium enoyl-ACP reductase (PfENR) cata- 2.33 mmol), and hydroxybenzotriazole (HOBt) (0.31 g,
lyzes the deterministic step of the elongation cycle and therefore, 2.29 mmol) was added anhydrous dimethyl formamide (DMF)
has emerged as an important drug target (9, 14–18).
(15 mL). The reaction mixture was stirred overnight at room tem-
Benzothiophene derivatives are heterocyclic aromatic compounds perature under nitrogen atmosphere till completion of the reaction.
which have found extensive pharmacologic applications. Benzothio- The reaction mixture was vacuum filtered to remove precipitated
phene biphenyl derivatives have been shown to have inhibitory ac- dicyclohexylurea. The filtrate was then evaporated under reduced
tivity against tyrosine phosphatase1B and antihyperglycaemic prop- pressure to give dark oil which was dissolved in ethyl acetate (20
erties (19). Raloxifene and arzoxifene are benzothiophene deriva- mL) and then refiltered. The filtrate was vacuum evaporated and
tives which act as selective estrogen receptor modulators of clinical the oily residue was purified by column chromatography (30%
value in postmenopausal osteoporosis, treatment of breast cancer, ethyl acetate: hexane). To prevent the product from crystallization
and potentially in hormone replacement therapy (20, 21). Benzothio- in the column, a short column was run under pressure to yield the
phene class of compounds act as antagonist to retinoid X receptors, target compounds (Scheme 1).
activator of a TRPV4 (Transient Receptor Potential Vallinoid sub-
type IV) channel (22) and also have antitumor properties (23).
N-Benzyl-3-bromobenzo[b]thiophene-2-carboxamide(4). Crystal-
PfENR which is an important antimalarial drug target is line colorless solid (0.78 g, 98%). M.p. 115–117 8C. Electron
effectively inhibited by bromo-benzothiphene carboxamide spray mass spectrometry (ES-MS) m/z: Calcd for C16H12BrNOS:
1
compounds. Hence, these compounds hold promise for the de- 346.24 [M1]; Obsd 346. H NMR (CDCl3, 300 MHz): 4.72 (d,
velopment of potent antimalarials.
2H, CH2), 7.31–7.45 (m, 5H, ArH benzyl), 7.48 (m, 2H, ArH),
and 7.83 (dd, 2H, ArH). 13C NMR: 44.5, 106.6, 122.9, 124.7,
125.8, 127.9, 128.0, 129.1, 137.7, 138.6, 138.7, and 161.1.
CHEMISTRY
Syntheses were performed according to the generic pathway
depicted in Scheme 1. Bromobenzo[b]thiophene-2-carboxylic acid
[2] was coupled with different commercially available arylalkyl
amines to obtain the target compounds 4–9 using activated ester of
bromobenzo[b]thiophene-2-carboxylic acid (24). In syntheses,
compounds 6 and 7 were treated with allyl bromide at 60 8C for 48
h to obtain 3-bromo-N-(4-fluorobenzyl)-N-(prop-2-en-1-yl)-ben-
zo[b]thiophene-2-carboxamide 10 and 3-bromo-N-(prop-2-en-1-
yl)-N-[4-(trifluoromethyl)benzyl]-1-benzo[b]thiophene-2-carboxa-
mide 11, respectively (25). Purity of each compound was 97–99%.
3-Bromo-N-(4-methoxybenzyl)-benzo[b]thiophene-2-carboxami-
de(5). Crystalline colorless, solid (0.85 g, 3.30 mmol). Mp. 123–
124 8C. ES-MS m/z: Calcd for C17H14BrNO2S: 376.27 [M1]; Obsd
1
376.26. H NMR (CDCl3, 300 MHz): 3.80 (s, 3H, OMe), 4.65 (d,
2H, CH2), 6.91 (m, 2H), 7.34 (m, 2H), 7.46 (m, 2H, ArH), and 7.83
(d, 2H, ArH). 13C NMR: 44.0, 55.5, 106.6, 114.4, 122.9, 124.7,
125.7, 125.7, 129.4, 135.1, 138.7, 159.4, and 161.0.
3-Bromo-N-(4-fluorobenzyl)-benzo[b]thiophene-2-carboxami-
de(6). M.p. 120–125 8C. ES-MS m/z: Calcd for
1
C16H11BrNOSF; 364.23 [M1]; Obsd 364.20. H NMR (CDCl3,
300 MHz) 4.70 (d, 2H), 7.45 (m, 2H, ArH) 7.83 (dd, 2H, ArH)
7 (m, 2H), and 7.36 (m, 2H). 13C NMR 44.0, 106.6, 125.7,
124.7, 123.9, 124.5, 130, 138.7, 163.5, and 161.0.
SYNTHESIS OF COMPOUNDS
General
All solvents (synthetic grade) were purchased from Merck
Schuchardt and purified before use. Benzothiophene-2-carbox- 3-Bromo-N-[4-(trifluoromethyl)benzyl]-benzo[b]thiophene-2-car-
ylic acid, benzyl amine, 4-methoxy benzyl amine, 4 fluoroben- boxamide(7). Mp. 128–130 8C. ES-MS m/z: Calcd for
zyl amine, 4-(trifluoromethyl)-benzyl amine, 2-phenyl ethyl C17H11BrF3NOS 414.24 [M1], Obsd 414. 1H NMR (CDCl3,
amine, and 1-naphthalene-methyl amine were purchased from 300 MHz): 4.70 (d, 2H), 7.47 (m, 2H, ArH), 7.12 (m, 2H), and
Aldrich. Melting points were determined with the Barnstaed 7.38 (m, 2H). 13C NMR 44.0, 106.6, 125.7, 124.7, 123.9, 124.5,
electrothermal apparatus and are uncorrected. Mass analysis 130, 138.7, and 161.0.

INHIBITORS OF PFENR
1103
Scheme 1. Synthesis of benzothiophene arylalkylamide derivatives. (i) Anhyd DMF, arylalkyl amine, DCC, HOBt, overnight (stir-
ring under N2 atmosphere at RT) (ii) Anhyd. THF, NaH/60 8C/30 min, CH2¼¼CHCH2Br, 48 h (heat with stirring). Conditions: (i)
Br2/CH3COONa, (ii) SOCl2/Toluene/Pyridine/3 h, (iii) activated ester of compound 2 and arylalkyl amines, and (iv) allyl bromide.
3-Bromo-N-(2-phenylethyl)-benzo[b]thiophene-2-carboxamide
General Procedure for Synthesis of 10 and 11. To a solution
(8). Colorless solid. ES-MS m/z: Calcd for C17H14BrNOS of 06 or 07 (1 mmol) in dry THF was added NaH and heated at
360.27 [M1]; Obsd 360. 1H NMR (CDCl3, 300 MHz): 3.5 60 8C for 30 min. Allyl bromide (1.2 mmol) was added to the
(d, 2H), 3.2 (d, 2H), 7.47 (m, 2H, ArH) 7.12 (m, 2H), and 7.38 resulting mixture and heated at 70 8C for 24 h. NaH and allyl
(m, 2H).13C NMR 42.9, 35.5, 106.6, 125.7, 124.7, 123.9, 124.5, bromide were further added to the reaction mass and maintained
130, 138.7, and 161.0.
at 70 8C for another 24 h. The solvent was vacuum evaporated,
and the residue was extracted with diethyl ether, washed with
3-Bromo-N-(naphthalen-1-ylmethyl)-benzo[b]thiophene-2-car- brine, dried and evaporated under reduced pressure. The residue
boxamide(9). Colorless solid. ES-MS m/z: Calcd for was purified by column chromatography (2% EtOAc-hexane;
1
C20H14BrNOS 396.30 [M1]; Obsd 396. H NMR (CDCl3, 300 Scheme 1)
MHz): 4.9 (d, 2H), 7.47 (m, 2H, ArH), 7.12 (m, 2H), 7.38 (m,
2H), 7.1 (m, 2H), 7.3 (m, 2H), and 7.6 (s, 1H). 13C NMR (75 3-Bromo-N-(4-fluorobenzyl)-N-(prop-2-en-1-yl)-benzo[b]thio-
MHz): 44.0, 125.7, 124.7, 123.9, 124.5, 127.4, 127.5, 128.3, phene-2-carboxamide (10). Colorless solid. ES-MS m/z: Calcd
1
128.2, 130, 138.7, and 161.0.
for C19H15BrFNOS 404.30 [M1]; Obsd 403.0. H NMR (CDCl3,

1104
BANERJEE ET AL.
300 MHz): 4.70 (d, 2H), 5.18 (d, 2H), 7.45 (m, 2H, ArH), 7.83 checked by SDS-PAGE. Purified enzyme was stored at 220 8C at
(2H, ArH), 7 (m, 2H), and 7.36 (m, 2H). 13C NMR (75 MHz): a concentration of 3 mg/mL till further use. PfFabB/F, PfFabG,
58.0, 106.6, 125.7, 124.7, 123.9, 124.5, 130, 138.7, 163.5, 136, and PfFabZ were purified and assayed as described earlier (30–32).
137.5, and 161.0.
Assay of PfFAS Enzymes
Enzyme assays and inhibition studies were performed on a
3-Bromo-N-(prop-2-en-1-yl)-N-[4-(trifluoromethyl)benzyl]-1-ben-
zo[b]thiophene- 2-carboxamide (11). Colorless solid. ES-MS m/
UV-Vis spectrophotometer (Shimadzu UV 2450) at 25 8C in 20
mM Tris/HCl and 150 mM NaCl, pH 7.4 (33). The standard reac-
1
z: Calcd for C20H15BrF3NOS 454.30 [M1]; Obsd 454.4. H NMR
(CDCl3, 300 MHz): 4.70 (d, 2H), 5.18 (s, 2H), 7.47 (m, 2H, ArH)
tion mixture (100 lL) contained 200 lM crotonoyl-CoA, 100 lM
NADH and 30 nM PfENR. All the inhibitors were dissolved in
DMSO. 1% DMSO was used in the standard reaction mixture.
7.12 (m, 2H), and 7.38 (m, 2H). 13C NMR 58.0, 106.6, 125.7,
124.7, 123.9, 124.5, 130, 136.4, 138.7, 142.2, and 161.0.
The reaction proceeds by reduction of crotonoyl-CoA to butyryl-
CoA with the oxidation of NADH to NAD1 which is monitored
at 340 nm (eM340 5 6220 M21 cm21). PfFabB/F, PfFabG, and
EXPERIMENTAL
Materials for PfENR Preparation and Enzyme Assay
Media components were purchased from Hi-media (Delhi,
India). b-NADH, crotonoyl CoA, imidazole, dimethylsulphonyl
oxide (DMSO), and sodium dodecyl sulphate/polyacrylamide
gel electrophoresis (SDS/PAGE) reagents were bought from
Sigma. All other chemicals were of analytical grade.
PfFabZ enzymes were assayed according to refs. 30–32.
Purification and Assay of Aldose Reductase
Recombinant human aldose reductase was expressed, puri-
fied, and assayed as described in refs. 34 and 35. The purified
enzyme was assayed spectrophotometrically as described in
detail in supporting information. Decrease in the absorption of
NADPH at 340 nm was measured over a period of 4 min with
DL-glyceraldehyde as the substrate. Inhibition of enzyme activ-
ity was monitored in the presence of compounds 6 and 7.
Preparation of the Receptor and Ligand Molecules
The crystal structure of PfENR submitted to protein database
(PDB) (PDB id: 1UH5) by Pidugu et al. (26) was used for dock-
ing simulations. Hydrogens were added and energy-minimized
using molecular modelling program Molecular Operating Envi-
ronment (MOE) (27). The three-dimensional coordinates were
then converted to mol2 format with MMFF94 charges using the
MOE suite of programs. Receptor was prepared for docking by
Protonate three dimensional Macromolecular Protonation State
Assignment tool part of MOE suite and partial charge of the re-
ceptor were fixed. Ligand molecules were prepared using MOE-
builder tool, a part of MOE suite and were subjected to energy
minimization to a gradient of 0.0001 using dielectric constant of
1 and partial charges were calculated by MMF94x force field.
Docking experiments were executed using molecular docking
structure-based design tool, a part of MOE suite 2009.10. The
compounds were docked by default nonstochastic Triangular
Matcher Placement method using the London dG scoring, fol-
lowed by force field refinement using default configurations of
docking simulations. Each ligand was docked in the receptor pro-
tein and top 100 conformations were retained. Docked complex
with least energy was chosen for ligand protein contact analysis.
The ligand receptor complex with compounds 6 and 7 along
with NAD were subjected to 15 cycles of energy minimization.
These ligand receptor protein complexes were further subjected
to LPC/CSU software for detailed analysis of the interatomic
contacts (Supporting Information Tables 1–3) (28).
Inhibition Studies of Alcohol Dehydrogenase
The activity of alcohol dehydrogenase (ADH) was measured
as described previously (36). The assay mixture in 1 mL con-
tained 76 mM sodium pyrophosphate-22 mM glycine buffer pH
8.0, 2.0 mM NA, 0.60 mM ethanol, and 0.01 lM alcohol dehy-
drogenase (Cat no. A-3263; Sigma). Appropriate blanks were
used for corrections. The assay mixture was incubated at 37 8C
and initiated by the addition of NAD at 37 8C. The change in the
absorbance at 340 nm because of NAD reduction was followed in
a Lamda35 spectrophotometer (Perkin–Elmer, Shelton).
Determination of IC50 Value for PfFAS Enzymes
IC50 values of the bromo-benzothiophene carboxamide class
of compounds were determined for PfFAS enzymes and aldose
reductase by plotting the percent activity of PfFAS versus loga-
rithm of the concentration of these compounds and the data
were analyzed by nonlinear regression method (37, 38).
Calculation of Dissociation Constants (Ki)
Ki of compound 6 was calculated with respect to NADH and
crotonoyl-CoA in separate experiments. With respect to NADH,
data were collected against two fixed concentrations of NADH
(100 lM and 200 lM) while varying the concentration of 6
from 0 to 750 nM keeping crotonoyl-CoA concentration fixed
Overexpression and Purification of PfFAS Enzymes
PfENR was overexpressed in E. coliBL-21(DE3) cells accord- at 200 lM. For Ki with respect to crotonoyl-CoA, data were
ing to the earlier published protocol with the only difference that collected against two fixed concentrations of crotonoyl-CoA
all buffers contained 10% glycerol (29). Purity of the enzyme was (100 lM and 200 lM) and concentration of 6 was varied from

INHIBITORS OF PFENR
1105
Figure 1. Compounds (a) 6 and (b) 7 docked with PfENR-NAD1 complex. The inhibitors are shown in ‘‘Balls and Sticks.’’ NAD
and active-site residues are shown in sticks. Figures were generated using MOE v208.10.
0 to 750 nM, whereas NADH was kept constant. Data were an-
alyzed by Dixon plot (39).
Potency Assays
Potency assays for inhibition of PfENR was designed as
described earlier (33). The activity of purified PfENR was
inhibited by addition of 6 (IC50 value) with and without prein-
cubation time. Addition of (10 lM) 6 to the above reaction was
shown to potentiate the inhibitory activity of 6. First, the control
reaction (a) was set as described earlier. The formation of
NAD1 was monitored at 340 nm. NAD1 accumulation was
observed to increase linearly with time. Reaction (b) contained
compound 6. Reaction (c) was set up with compound 6 but
with a preincubation time of 20 min. In reaction (d) the enzyme
was preincubated with both NAD1 and compound 6.
Determination of Association (k5 or kon) and
Dissociation Rate Constants (k6 or koff) of
Compounds 6 and 7 for PfENR
Figure 2. (a, b) Inhibition of PfENR at various concentration
The association or isomerization rate constant (k5) of com-
(0–1 lM) of compounds. The percentage activity was calculated
pounds 6 and 7 with PfENR was determined separately by mon-
itoring the onset of inhibition in the enzyme reactions. In the
reaction mixture compounds 6 and 7 were added (0–750 nM)
sequentially and the formation of NAD1 was plotted against
time by nonlinear regression method using Eq. (2a) discussed
later to get kobs for each concentration of compounds 6 and 7
used. The values of kobs were fitted to Eq. (3) to get Ki, k5, and
k6 values. The hyperbolic curve obtained clearly depicts the
slow tight binding nature of inhibition (40, 41).
from residual activity and was plotted against log concentration.
(a) Compound 6 and (b) compound 7. (c) Inhibition kinetics of
compound 6 with respect to NADH. The effect of NADH on in-
hibition by compound 6 was determined using Dixon plot. 30
nM PfENR was assayed in the presence of 200 lM crotonoyl
CoA and at two concentrations of NADH, 100 lM [~], 150
lM [l] with various concentration of compound 6. Ki of com-
pound 6 was calculated from the X-intercept using equation for
competitive kinetics. Each data point indicates three different
data sets and the error bar indicates the standard deviation of
the data. (d) Inhibition of PfENR by compound 6 with respect
Evaluation of the Kinetic Data
Initial rate constants were determined using Dixon plot to crotonoyl CoA. PfENR was assayed at two fixed concentra-
assuming the reaction to be competitive for compound 6 against tions 200 lM [l] and 300 lM [~] of crotonoyl CoA in the
NADH and uncompetitive for compound 7 against crotonoyl- presence of 100 lM of NADH and various concentration of
CoA. The data were plotted using the following Dixon equa- compound 6. Ki was calculated from the X-intercept of Dixon
tions for competitive [Eq. (1a); ref. 39] and uncompetitive plot assuming uncompetitive kinetics. Each data point repre-
kinetics [Eq. (1b); ref. 39], respectively.
sents the three independent sets of experiments.

1106
BANERJEE ET AL.
mechanisms, it is presumed that the slow binding inhibition
step is reversible.
Table 1
Inhibitory potencies of bromobenzothiphene derivatives
against purified PfENR
k3
E þ I $ E:I
ðMechanism 1Þ
ðMechanism 2Þ
Compound
Triclosan
Structure
Inhibition of PfENR (IC50lM)
0.089 6 0.005
k4
k3
k5
E þ I $ E:I $ E:Iꢁ
k4
k6
2
4
5
1.68 6 0.045
0.501 6 0.033
31.99 6 0.23
The biphasic progress curves which are typical for slow tight
binding inhibition were fitted to Eq. (2a) (40, 41) using nonlin-
ear least squares fitting procedure. Corrections were made for
the variation of the steady-state velocity with the inhibitor con-
centrations using Eqs. (2b) and (2c) (40, 41) as described earlier
by Morrison and Walsh (40, 41).
ðvo ꢂ vsÞ½1 ꢂ expðꢂkobs3tÞꢀ
P ¼ vs3t þ
þ C
(2a)
kobs
6
7
8
9
0.115 6 0.012
0.463 6 0.01
54.98 6 0.42
52.56 6 0.33
k7SQ
vs ¼
(2b)
(2c)
2ðKm þ SÞ
1=2
Q ¼ ½ðKi0 þ It ꢂ EtÞ þ 4Ki0Etꢀ ꢂ ðKi0 þ It ꢂ EtÞ
k7, is the rate constant of the product formation
k7
k1
[E þ S $ ES $ E þ P].
k2
The relationship between Ki, k5, k6, and kobs is described in
Eq. (3).
0
@
1
A
1
Ki
ꢀ
ꢁ
ꢀ ꢁ
kobs ¼ k6 þ k5
½Sꢀ
Km
½Iꢀ
1 þ
þ
Ki
10
11
51.98 6 0.43
52.48 6 0.22
RESULTS AND DISCUSSION
The elongation cycle of type II FAS involves four successive
reactions: decarboxylative condensation to condense the growing
acyl chain with malonyl-ACP by b-ketoacyl-ACP synthase
(FabF), NADPH dependent reduction by b-ketoacyl-ACP reduc-
tase (FabG), dehydration by b-hydroxyacyl-ACP dehydratase
(FabA or FabZ), and NADH dependent reduction by enoyl-ACP
reductase (FabI or PfENR) (9, 14–18). All these enzymes have
thioester substrates. Bromo-benzothiophene carboxamide deriva-
tives have amide linkage in the midst of nonpolar moieties on
either side which mimics the thioester linkage of the substrate.
Therefore, the bromo-benzothiophene derivatives were tested
against purified FabF, FabG, FabZ, and FabI. The compounds did
not inhibit any other enzymes of the elongation cycle of malaria
parasites fatty acid synthesis, namely FabF, FabG, and FabZ
excepting PfENR. Aldose reductase an NADPH dependent oxi-
1
km3½Iꢀ
1
ꢀ
ꢁ
¼
þ
(1a)
(1b)
Km
½Sꢀ
v
Vmax3Ki3½Sꢀ
Vmax 1 þ
1
½Iꢀ
1
ꢀ
ꢁ
¼
þ
Vmax3Ki
Km
½Sꢀ
v
Vmax 1 þ
Time dependent reversible inhibition can be described by doreductase or alcohol dehydrogenase an NAD dependent
any of the two mechanisms described later (39). In both the enzyme were not inhibited by compound 6, the best inhibitor of

INHIBITORS OF PFENR
1107
Table 2
Dissociation constant, isomerization rate constant, dissociation rate constant of compounds 6 and 7 against purified PfENR
Dissociation
Isomerization rate
constant (k5 3 1022) (s21
Dissociation rate constant
(k6 3 1023) (s21
)
Compounds
constant (Ki) nM
)
6
7
48.87
86.1
1.19 6 0.045
1.65 6 0.023
1 6 0.0004
6.9 6 0.0003
PfENR. These compounds thus inhibit FabI in specific manner. are predominantly van der Waals interactions. Compound 6 also
Other features of PfENR inhibition like (1) at least one of the has extensive van der Waal’s interaction with NAD1. Benzyl
rings in the inhibitors participate in P–P stacking interactions ring of compound 6 makes P–P stacking interaction with nico-
with the nicotinamide ring of the cofactor NADH and (2) the tinamide ring of NAD1. Its carbonyl group interacts with N7
compound forms a hydrogen bond with the active site residue, ty- group of NAD1. Compound 7 has six hydrogen bonding inter-
rosine, are also satisfied by these compounds (26, 29). Docking actions, 29 hydrophobic interactions, 18 aromatic–aromatic
studies discussed later further revealed optimum interactions interactions and five hydrophobic–hydrophilic interactions (Fig.
between these compounds and PfENR.
1b; Supporting Information Tables 1 and 3). There are 99 other
To improve the efficacy of these compounds, bromo-benzo- interactions mostly van der Waals type. Compound 7 also has
thiophene carboxamide core was further derivatized with benzyl extensive van der Waal’s interaction with NAD1 as well as P–
group attached via biodegradable amide (CONH) linkage. Incor- P stacking interaction with nicotinamide ring of NAD1.
poration of amide linkage improved the efficacy of the com-
pound (compound 2) marginally. Toward this end, benzyl group
was further derivatized with various substituents like aromatic
groups, halogens, and alkyl groups. We observed that fluoro
derivatives (compounds 4, 6, and 7) have significant improve-
ment in their efficacy compared with other derivatives.
Kinetics of the Inhibition of PfFAS Enzymes
PfENR activity was determined using standard assay
described earlier (37). The IC50 of the three best compounds
were in nanomolar range and the remaining compounds were
active at micromolar concentrations (Figs. 2a and 2b, Table 1).
Compound 6 (IC50 5 0.115 6 0.12 lM) was found to be most
potent. The IC50 of other two potent compounds were also in
Docking of Benzothiophene Derivatives with PfENR
Docked conformations of bromo-benzothiophene carboxa-
mide derivatives to PfENR reveal that these molecules occupy
the hydrophobic pocket composed of Tyr 267, Ala 272, Tyr277,
Gly 313, Pro 314, Phe 368, and Ile 369. Benzyl group is
observed to be surrounded by substrate binding loop residues
Ala 319, Ala 320, and Ile 323 and another loop from Ala 217
to Val 222. Amide group mimics the thioester of the substrate.
Compound 6 has two hydrogen bonding interactions, 28 hydro-
phobic interactions, 16 aromatic–aromatic interactions, and 12
hydrophobic–hydrophilic interactions (Fig. 1a; Supporting Infor-
mation Tables 1 and 2). There are 89 other interactions which
Table 3
Inhibitory potencies of compound 6 against purified PfFAS
enzymes
Figure 3. Potency assay of compound 6 for PfENR. The po-
tency assay of compound 6 shows the slow onset of inhibition
of PfENR by 6. Curve ‘‘a’’ is control reaction without com-
pound 6 but contained 1% DMSO, curve ‘‘b’’ is inhibition reac-
tion in the presence of 115 nM of compound 6, curve ‘‘c’’
shows further potentiation of inhibition when compound 6 was
pre incubated with 35 nM of PfENR for 20 min, curve ‘‘d’’ is
the inhibition reaction by compound 6 when 10 lM of NAD1
was added to the preincubated mixture. The y-axis represents
the accumulation of product NAD1 in mM concentration with
respect to time (in seconds, x-axis).
Enzymes
Inhibition at 10 M
PfFabF
PfFabG
PfFabZ
PfFabI
ALR2
ADH
No inhibition
No inhibition
No inhibition
99%
\1%
No inhibition

1108
BANERJEE ET AL.
increase in the accumulation of NAD1 (curve a), whereas when
the reaction was carried out in the presence of compound 6,
accumulation of product decreased (curve b). The accumulation
of product further decreased when compound 6 was preincu-
bated with the enzyme (curve c). On addition of NAD1 to this
reaction the rate of product accumulation further decreased
(curve d). This also indicated that these are slow tight binding
inhibitors of PfENR.
Detailed Analysis of the Progress Curves of
Bromo-benzothiophene Carboxamide Derived
Inhibitors for PfENR
Progress curves for inhibition by varying concentrations of
compounds 6 and 7 were examined in detail (Figs. 4a and
4b). From the progress curve analyses, it was adjudged that
for each concentration of the inhibitors the initial and the
steady state velocity decreased exponentially with time. At
higher inhibitor concentration, steady state is reached rapidly
with a decrease in steady state velocity (vs). It indicates that
initially a loose complex of inhibitor, NAD1, and PfENR is
formed which slowly isomerises into a more stable tight
complex.
Figure 4. (a, b) Progress curves of compounds. For each con-
centration of compounds, progress curve was generated using
Eq. (2a). In the control, standard reaction was set (as described
in Experimental Section), and contained 1% DMSO apart from
other constituents. Compounds were added in the reaction mix-
tures at various concentration (0–750 nM) as indicated in figure.
The y-axis represents the accumulation of product NAD1 in
mM concentration with respect to time (in sec, x-axis). (a)
Compound 6 and (b) compound 7. (c, d) Dependence of PfENR
inhibition on different concentrations of compounds. The initial
inhibition rate constant (kobs) (y-axis) for each compound con-
centration added was calculated from the progress curves using
Eq. (2a). The kobs thus calculated were fitted to Eq. (3) with
respect to respective concentrations of each compound and the
best fit gave hyperbolic curve demonstrating two phase inhibi-
tion mechanism. The error bars represents the standard devia-
tion of the data. (c) Compound 6 and (d) compound 7.
Progress curves were analyzed using Eq. (3a) from which a
series of Kobs values were obtained for each concentration of in-
hibitor. The determined Kobs values were plotted against respec-
tive concentrations of compounds 6 and 7 which resulted in
hyperbolic curve (Figs. 4c and 4d). Hyperbolic curve indicated
biphasic binding of inhibitors again reflecting slow tight binding
behavior of bromo-benzothiophene carboxamide derived inhibi-
tors of PfENR (40, 41).
CONCLUSIONS
De novo fatty acid synthesis in the malaria parasites either
provide unique fatty acids or its role is to augment levels of sal-
vaged fatty acids (from host) to meet the huge demand during
their growth phase. In either case, disruption of the de novo
synthesis in parasites, by inhibitor(s), is an attractive target.
Bromo-benzothiophene carboxamide compounds specifically in-
hibit PfENR and not the other enzymes of FASII elongation
module, NADPH dependent aldose reductase, or NAD alcohol
dehydrogenase. Compounds 6 and 7 inhibited PfENR with an
IC50 of 115 nM and 463 nM, respectively, proving that PfENR
is a key target of these compounds and were found to be slow
tight binding inhibitors of PfENR. Hence these compounds hold
promise to be significant antimalarials.
the nanomolar range (Compounds 7 5 0.463 6 0.1 lM, 2 5
0.51 6 0.33 lM). Ki values for the best inhibitor, compound 6
were determined individually against cofactor NADH and sub-
strate crotonoyl CoA. It gave competitive kinetics with NADH
and uncompetitive against crotonoyl CoA (Figs. 2c and 2d).
The Ki values are indicated in Table 2. Compound 6 was shown
to have IC50 more than 100 lM against other purified FAS
enzymes (Table 3). Analysis of kinetic data showed that these
inhibitors compete with NADH for binding to PfENR.
ACKNOWLEDGEMENTS
The authors thank the Department of Biotechnology, Govern-
ment of India, for the grant to N.S. and A.S. as well as Centre
of Excellence grant to A.S. N.S. holds TATA Innovation fel-
Cofactor Potency Assays
NAD1 was shown to potentiate the binding of compound 6 lowship of the Department of Biotechnology, Government of
to PfENR (Fig. 3) (38). The control reaction showed a linear India. T.B. and S.K.S. thank the Council of Scientific and

INHIBITORS OF PFENR
1109
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