Design, synthesis and characterization of novel inhibitors against mycobacterial β-ketoacyl CoA reductase FabG4

Article abstract of DOI:10.1039/c3ob41676c

We report the design and synthesis of triazole-polyphenol hybrid compounds 1 and 2 as inhibitors of the FabG4 (Rv0242c) enzyme of Mycobacterium tuberculosis for the first time. A major advance in this field occurred only a couple of years ago with the X-ray crystal structure of FabG4, which has helped us to design these inhibitors by the computational fragment-based drug design (FBDD) approach. Compound 1 has shown competitive inhibition with an inhibition constant (K_i) value of 3.97 ± 0.02 μM. On the other hand, compound 2 has been found to be a mixed type inhibitor with a K_i value of 0.88 ± 0.01 μM. Thermodynamic analysis using isothermal titration calorimetry (ITC) reveals that both inhibitors bind at the NADH co-factor binding domain. Their MIC values, as determined by resazurin assay against M. smegmatis, indicated their good anti-mycobacterial properties. A preliminary structure-activity relationship (SAR) study supports the design of these inhibitors. These compounds may be possible candidates as lead compounds for alternate anti-tubercular drugs. All of the reductase enzymes of the Mycobacterium family have a similar ketoacyl reductase (KAR) domain. Hence, this work may be extrapolated to find structure-based inhibitors of other reductase enzymes. The Royal Society of Chemistry.

Full text of DOI:10.1039/c3ob41676c

Organic &
Biomolecular Chemistry
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Design, synthesis and characterization of novel
inhibitors against mycobacterial β-ketoacyl CoA
reductase FabG4†
Cite this: Org. Biomol. Chem., 2014,
12, 73
Deb Ranjan Banerjee,a Debajyoti Dutta,b Baisakhee Saha,b Sudipta Bhattacharyya,b
Kalyan Senapati,a Amit K. Das*b and Amit Basak*a
We report the design and synthesis of triazole-polyphenol hybrid compounds 1 and 2 as inhibitors of the
FabG4 (Rv0242c) enzyme of Mycobacterium tuberculosis for the first time. A major advance in this field
occurred only a couple of years ago with the X-ray crystal structure of FabG4, which has helped us to
design these inhibitors by the computational fragment-based drug design (FBDD) approach. Compound 1
has shown competitive inhibition with an inhibition constant (Ki) value of 3.97 0.02 μM. On the other
hand, compound 2 has been found to be a mixed type inhibitor with a Ki value of 0.88 0.01 μM. Ther-
modynamic analysis using isothermal titration calorimetry (ITC) reveals that both inhibitors bind at the
NADH co-factor binding domain. Their MIC values, as determined by resazurin assay against M. smegmatis,
indicated their good anti-mycobacterial properties. A preliminary structure–activity relationship (SAR)
study supports the design of these inhibitors. These compounds may be possible candidates as lead com-
pounds for alternate anti-tubercular drugs. All of the reductase enzymes of the Mycobacterium family
have a similar ketoacyl reductase (KAR) domain. Hence, this work may be extrapolated to find structure-
based inhibitors of other reductase enzymes.
Received 15th August 2013,
Accepted 19th September 2013
DOI: 10.1039/c3ob41676c
www.rsc.org/obc
Mycobacterium.4 FAS-I, which is involved in the de novo syn-
thesis of fatty acids, is a multi-domain enzyme. The FAS-II
Introduction
Finding a new target to fight an existing disease has always pathway is necessary for the synthesis of long chain fatty acids.
been at the center of research in medicinal chemistry. Tuber- These long-chain fatty acids are finally used in mycolic acid
culosis (TB) is one such existing disease which is still causing synthesis. Hence, the enzymes involved in FAS are attractive
problems for mankind.1 The World Health Organization targets for drug designing. FabG, a ketoacyl reductase, is one
(WHO) has estimated that about one-third of the world popu- of the major enzymes involved in FAS. The Mtb genome con-
lation is infected by its causative agent Mycobacterium tuber- tains five FabG genes,5 but only two, FabG1 (Rv1483) and
culosis (Mtb).2 The emergence of multi drug resistant (MDR) and FabG4 (Rv0242c), are conserved among the mycobacterial
extremely drug resistant (XDR) Mtb strains has complicated species. FabG1 is a well-known β-ketoacyl CoA reductase6 and
the scenario.3 First line TB drugs such as isoniazid, rifampicin, has been a good candidate for alternate drug discovery in the
pyraniazid, ethambutol and streptomycin have failed in recent last decade.7 FabG4 is the less explored gene, which has
TB cases, so it is important to find alternative drugs to fight recently been reported to be an essential and functional gene
against tuberculosis.
for bacterial growth, survival8 and fatty acid synthesis.9
M. tuberculosis possesses a lipid-rich cell envelope which is FabG4 may also have a role in the drug resistance of mycobac-
required for its survival within the host cell. The main com- terial species as it is over-expressed in sub-inhibitory concen-
ponent of this virtually impenetrable cell wall, mycolic acids, trations of Streptomycin.10 These reports in the last three years
is produced through fatty acid synthesis (FAS). Two fatty acid have made FabG4 a new attractive target to fight tuberculosis.
synthesis pathways, FAS-I and FAS-II, have been reported in
FabG4 is a high molecular weight ketoacyl reductase
(HMwFabG). The crystal structure of FabG4 shows the presence
of two distinct domains, domain I and II.11 Domain I is an
extra N-terminal domain, and domain II is a typical ‘ketoacyl
CoA reductase (KAR) domain’. FabG4 is a NADH-dependent
ketoacyl reductase, whereas FabG1 is a NADPH-dependent
ketoacyl reductase enzyme. The conserved catalytic tetrad
aIIT Kharagpur, Chemistry, Kharagpur, West Bengal, India.
E-mail: absk@chem.iitkgp.ernet.in
bIIT Kharagpur, Biotechnology, Kharagpur, West Bengal, India
†Electronic supplementary information (ESI) available. See DOI:
10.1039/c3ob41676c
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smegmatis. A preliminary structure activity relationship (SAR)
has been carried out to justify the design of the inhibitors.
Results and discussion
Design strategy
The recently-solved X-ray crystal structure of FabG4 with co-
factor NADH provides the way to design the first inhibitors
against this enzyme. We targeted the co-factor (NADH)-
binding domain for design purposes. Inhibitors which target
the NADH binding site can interact with the three subsites of
the coenzyme binding domain: the nicotinamide-binding
subsite (N-subsite), the adenosine-binding subsite (A-subsite)
and the pyrophosphate-binding subsite (P-subsite). For
Fig. 1 KAR domain of FabG4 with co-crystallized NADH and HXC. The
catalytic tetrad (Ser347, Tyr360, Lys364 and Asn319) and other crucial
amino acids are shown (left image). NADH accesses the active site from
the major portal; HXC accesses from the minor portal (right image).
Ser347, Tyr360, Lys364 and Asn319 constitute the active site structure-based design purposes, we followed the compu-
residues of the KAR domain. The active site is covered by loop tational fragment-based drug design (FBDD) strategy.13 In this
I and loop II. The structure of FabG4 co-crystallized with hexa-
approach, fragments and linkers were selected from a litera-
noyl-CoA (HXC) and NAD+ (PDB ID: 3V1U) suggests that the ture survey and merged to form a library. The selection of the
active site of FabG4 could be accessed from two different lead compounds was performed through computational
sides: the major portal and minor portal.12 The major portal is
docking studies. The advanced molecular grid-based docking
wide open and is accessed by the cofactor to bind with the program Autodock4.214 was used for the docking of a flexible
enzyme. The minor portal is narrower due to the helices of the ligand within a flexible protein.
dimeric interface and is accessed by the 4 phosphopan-
Epigallocatechin gallate and related plant polyphenols are
tetheine-bound fatty acyl substrates. The active site is covered known inhibitors of bacterial FabG.15 Hence, polyphenols were
from one side by two conserved loops, loop I and loop II. The selected as one of the components of the fragment-based
conserved NAG triad (Asn295, Ala296, Gly297) of loop I inter-
library. Moreover, in recent years, triazoles have received much
acts with the pyrophosphate section of the substrate and guides attention as a central core in medicinal chemistry, and
the cofactor towards the active site. Asp244 and Val268 have a especially in the synthesis of anti-tubercular agents.16 The 1,4-
crucial role to hold NADH at the major portal by interacting
triazole moiety can also be used as a conformationally con-
with the adenine section of the coenzyme. Asp244 and Arg223 strained bioisosteric replacement of the pyrophosphate linker
are the major factors behind NADH selectivity (Fig. 1).
of NADH.17 With this in mind, we selected a 1,4-triazole linker
for lead optimization. From the lead library, two novel 1,4-tria-
Based on the information about the overall structure and
specificity of the FabG4 enzyme, we have attempted to design zole linked polyphenols 1 and 2 were selected as possible
novel structure-based inhibitors of FabG4. The synthesis of the inhibitors of FabG4 on the basis of their docking score (Fig. 2).
designed compounds were carried out and their bio-activities
The docking study suggested that the binding of the 1,4-tria-
were measured though biochemical assays. The drug suscepti- zole moiety occurs at the pyrophosphate binding region
bilities of these inhibitors were tested against Mycobacterium (P-subsite), whereas the polyphenol fragments compete for the
Fig. 2 Selected 1,4-triazole linked polyphenol hybrids.
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N- and A-subsites of the NADH binding region. The detailed
For the synthesis of compounds 1 and 2, (−)-epicatechin
interactions from the docking studies are discussed later in and (+)-catechin were used as the starting materials respecti-
this report. To show the importance of fragments linked by the vely. To obtain the alkyne component for the click reaction,
1,4-triazole moiety, two more hybrids, 3 and 4, were designed tetrabenzyl catechin or epicatechin was propargylated to 3-O-
to draw preliminary structure–activity relationships.
propargyl tetrabenzyl catechin or epicatechin. The azide com-
ponent was made from gallic acid. Thus, tri-O-benzyl gallic
acid was converted to the respective alcohol by NaBH4
reduction in ethanol. The SN2 replacement of the benzylic –OH
by bromide, followed by azide, led to the other component for
the click reaction. The click reaction was performed in the
presence of Cu(I) made in situ by the reduction of CuSO4 with
sodium ascorbate. Debenzylation with H2 in the presence
of Pearlman’s catalyst resulted in the final compounds
(Scheme 1).
Compounds 3 and 4 were synthesized via a similar pro-
cedure using a click protocol (Scheme 2). m-Cresol was
selected as the starting material to obtain compound 3. For
the synthesis of compound 4, a click reaction was carried
out between benzyl azide and 3-O-propargyl tetrabenzyl
Synthesis
The methodology to obtain triazole derivatives was Huisgen’s
1,3-dipolar cycloaddition of azides and alkynes (click proto-
col).18 A click reaction was performed in the presence of Cu(I),
made in situ by the reduction of CuSO4 with sodium ascorbate.
As expected, the regioselectivity of the click reaction was con-
trolled by the Cu(I) catalyst, and only 1,4-regioisomers were
formed.19 The respective azide and alkyne counterparts were
synthesized from relatively cheaper and easily available start-
ing materials. The benzyl ether was chosen as the protecting
group of the polyphenol because of its easy deprotection
under neutral conditions which will avert the possible race-
mization of the polyphenol at the C-2 position.
Scheme 1 Synthesis of compounds 1 and 2. Reagents and conditions: (i) BnBr, K2CO3, 24 h; (ii) propargyl bromide, NaH, dry THF, 0 °C to RT, 8 h–
10 h; (iii) conc. H2SO4 (cat.), dry MeOH, reflux, 14 h; (iv) BnBr, K2CO3, dry DMF, 24 h; (v) LAH, dry THF, 0 °C, 4 h; (vi) PBr3, dry DCM, 0 °C, 20 min;
(vii) NaN3, DMF, 12 h; (viii) CuSO4–sodium ascorbate, MeCN–H2O (1 : 1), 36 h–48 h; (ix) 20% Pd(OH)2–C, THF–MeOH–H2O (20 : 1 : 0.5), 7 h.
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Scheme 2 Synthesis of compounds 3 and 4 for SAR. Reagents and conditions: (i) propargyl bromide, NaH, THF, 0 °C to RT, 48 h; (ii) compound 13,
CuSO4–sodium ascorbate, MeCN–H2O (1 : 1), 24 h; (iii) Pd(OH)2–C, THF–MeOH–H2O (20 : 1 : 0.5), H2 (2 bar), 5 h; (iv) NaN3, dry DMF, 12 h; (v) com-
pound 6, CuSO4–sodium ascorbate, MeCN–H2O, 4 h; (vi) Pd(OH)2–C, THF–MeOH–H2O (20 : 1 : 0.5), H2 (2.5 bar), 7 h.
epicatechin. Finally, debenzylation using 20% Pd(OH)2–C at hand, compound 2 was found to be a mixed type inhibitor
high hydrogen pressure afforded the final compounds.
All of the final compounds were purified by repeated pre-
cipitation from methanol–ether and characterized by 1H and
13C NMR, and mass spectrometry. The purity of these com-
pounds was determined through reverse-phase analytical
HPLC (see ESI†).
with a Ki value of 0.88 0.01 μM (Fig. 4).
Thermodynamic analysis
The thermodynamic parameters for inhibition were obtained
from isothermal titration calorimetry (ITC). The ITC studies of
FabG4 with compound 1, compound 2 and NADH were carried
out at 25 °C. The FabG4–NADH titration was first performed as
a control experiment because NADH was used as a substrate in
the inhibition studies. The binding curve of the FabG4–NADH
Inhibition kinetics
To evaluate the inhibitory effects of our compounds, the inhi- titration nicely fits to the ‘sequential binding’ mode, with the
bition kinetics were carried out at 25 °C. The decrease in the number of binding sites (N) equal to 2 (Fig. 5A). This sequen-
absorbance value at 340 nM due to the conversion of NADH to tial nature of binding is reasonable because FabG4 exists as an
NAD+ was monitored in this assay. Half maximal inhibitory inseparable homo-dimer in solution. NADH binding to one
concentration (IC50) values and modes of inhibition were eval- monomeric unit regulates the binding of a second NADH to
uated kinetically. (−)-Epicatechin and (+)-catechin, the starting the second monomer, and this is co-operative in nature. This
materials of compounds 1 and 2 respectively, did not inhibit phenomenon is similar to other known FabGs where the
FabG4 up to a 100 μM concentration. However, our designed oligomeric interface is responsible for cooperativity.20 NADH
and synthesized compounds 1 and 2 showed promising inhi- binding to both the monomers is spontaneous, as suggested
bition against Mtb FabG4. Both the compounds inhibited by the ΔG values obtained from ITC (Table 1). The first
FabG4 at low micromolar concentrations. The IC50 values of binding constant (K1) is higher than the second binding con-
compounds 1 and 2 are 44.7 μM and 34.9 μM respectively stant (K2), which indicates negative co-operativity.
(Fig. 3). Compound 1 showed competitive inhibition with an
Compound 1, which has been found to be a competitive
inhibition constant (Ki) value of 3.97 0.02 μM. On the other inhibitor with respect to NADH, showed a similar binding
Fig. 3 Dose–response plots to determine the IC50 of compounds 1 (A) and 2 (B).
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Fig. 4 The mode of inhibition on Mtb FabG4. (A) Compound 1 is a competitive type inhibitor of FabG4 with respect to NADH. The double-reciprocal
plot of 1/v versus 1/[NADH] at different concentrations of 1 intercepts the 1/v axis. (B) Compound 2 is a mixed type inhibitor of FabG4 with respect to
NADH. The double-reciprocal plot of 1/v versus 1/[NADH] at different concentrations of 2 intercepts to the left of the 1/v axis and above the
1/[NADH] axis, indicating that compound 2 is a mixed type inhibitor for NADH.
Fig. 5 Calorimetric titration raw data (upper panel) and resulting integrated binding isotherms with the best fit to the sequential binding model
(lower panel) of NADH (A), compound 1 (B) and compound 2 (C) with FabG4 at 25 °C.
Table 1 Mode of binding, binding constants and thermodynamic parameters from ITC titrations; K1, ΔH1, ΔS1, ΔG1 and K2, ΔH2, ΔS2, ΔG2 represent
the parameters of NADH, compound 1 or compound 2 at the first and second monomers of the FabG4 dimer respectively
Thermodynamic parameters
Ligand
Mode of binding
Binding constant K (mol−1
)
ΔH (kcal mol−1
)
ΔS (cal mol−1 K−1
)
ΔG (kcal mol−1
)
NADH
Sequential binding, with number of
binding sites (N) = 2
Sequential binding, with number of
binding sites (N) = 2
Sequential binding, with number of
binding sites (N) = 2
K1 = 5.43 × 105
K2 = 6.13 × 104
K1 = 2.76 × 105
K2 = 1.17 × 103
K1 = 1.36 × 105
K2 = 1.10 × 104
ΔH1 = −15.4
ΔH2 = −15.8
ΔH1 = −14.3
ΔS1 = −25.6
ΔG1 = −7.8
ΔG2 = −6.6
ΔG1 = −7.4
ΔG2 = −4.5
ΔG1 = −6.9
ΔG2 = −6.4
ΔS2 = −31.2
Compound 1
Compound 2
ΔS1 = −23.2
ΔH2 = −1.1 × 103
ΔH1 = −8.5
ΔS2 = −3.7 × 103
ΔS1 = −5.2
ΔH2 = −5.1 × 102
ΔS2 = −1.7 × 103
nature. The calorimetry of compound 1 with FabG4 revealed previous finding that compound 1 competes with NADH for
that it also binds sequentially to each monomer, as in the case the same binding sites. The binding of compound 1 is spon-
of NADH (Fig. 5B). Thus, the calorimetric data supports our taneous, with a negative ΔG (Table 1). The first binding of
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compound 1 is strong (K1 = 2.76 × 105 M−1), and the para- and Thr299 at loop I at the P-subsite. It interferes with the
meters indicate interactions via good hydrogen bonding and nicotinamide binding region (N-subsite) of NADH by making
conformational changes.21 The second binding constant is low two H-bonds with Ser346. Compound 1 also interacts with the
(K2 = 1.17 × 103 M−1), which is attributed to the high negative catalytic tetrad directly via H-bonding with the active site
co-operativity.
residue Lys364. Moreover it corroborates our kinetic and
The FabG4–compound 2 titration also fits to the ‘sequential thermodynamic findings that compound 1 binds at the NADH
binding’ mode with two bindings (Fig. 5C). The binding of binding domain via hydrogen bonds and acts as a competitive
compound 2 at the NADH binding pockets of both monomers inhibitor.
is spontaneous and comparable to compound 1 (Table 1). As
Compound 2 also binds at the major portal, though it does
evidenced from the thermodynamic parameters obtained from not compete with all of the binding sites of the NADH binding
ITC, compound 1 binds more strongly than compound 2 at the region. According to the docking study, the binding energy of
first monomeric NADH binding site. In the case of the second compound 2 (−5.00 kcal mol−1) is lower than for compound 1
binding, the extent of negative co-operativity is lower than (−6.34 kcal mol−1). This supports the previous thermodynamic
for compound 1, as the second binding of compound 2 is findings that compound 2 also binds at the NADH binding
relatively stronger than the second binding of compound 1.
domain, but the binding is relatively weaker than for com-
The binding of NADH and the inhibitors to the major pound 1. The binding mode of compound 2 shows that it also
portal of FabG4 is mainly governed by H-bonding with Asp244, accesses the active site through loop I via H-bonding with
Arg223, Asn295 and the catalytic tetrad residues. Such types of Ser347 of the catalytic tetrad. It interferes at the N-subsite of
hydrophilic interactions are also supported by the negative NADH, binding by H-bonds with Ser346 and Gly391. Com-
values of both ΔH and ΔS obtained from the ITC experiments pound 2 shows H-bonding with Arg300 of loop I. However,
(Table 1). The negative ΔH and ΔS indicates the contribution unlike compound 1, the catechin subunit of compound 2 does
of strong H-bonding and conformational changes respectively.
not show any interaction at the A-subsite or P-subsite of the
NADH binding region, whereas the gallol subunit forms
H-bonds with Gly391, one of the conserved residues in the
α-helical sub-domain which provides the flexibility of the sub-
domain to access the substrate acetoacetyl CoA.12 Thus, these
findings indicate that compound 2 can interfere with the
enzyme–substrate complex, as well as the free enzyme, result-
ing in the mixed type of inhibition (Fig. 6).
Docking studies
Results from docking studies support the experimental find-
ings and provide necessary theoretical insight into the binding
mode of the compounds. Docking studies can not explain the
aspects of sequential binding as it was performed between
one ligand and one monomer file. Here, the docking studies
provides the binding interaction of one monomer with the
inhibitor.
Minimum inhibitory concentration (MIC assay)
Compound 1 interacts with several residues via hydrogen The drug MICs for Mycobacterium species were determined by
bonding and fully competes with the NADH binding region an aerobic resazurin microplate reduction assay (REMA).22
(major portal). The docking study shows that compound 1 Mycobacterium smegmatis was used as a model organism
interacts with Leu266, Gly220 and Val268 at the A-subsite. It because approximately two thousand of its proteins share
also shows hydrogen bonding with Gly297 of the NAG triad homology with the pathogen M. tuberculosis. Additionally,
Fig. 6 Docking of compounds 1 (A) and 2 (B) in FabG4, showing the interacting amino acid residues. NADH is shown for comparison. Compound 1
competes at every binding position with the coenzyme. Compound 2 mainly interacts with the active site and loop I. The compounds are shown in
atom colors; NADH is shown in magenta. Hydrogen bonds are shown as yellow dotted lines. The images are made with PyMOL.
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it shares similarities with M. tuberculosis in possessing the
The inhibition studies of 3 and 4 were carried out under
unusual cell wall structure. The functional complementation identical conditions and compared with the original inhibitors
of the essential gene, fabG1, of Mycobacterium tuberculosis by (Table 2). Compound 3 did not inhibit FabG4 up to a 400 µM
Mycobacterium smegmatis fabG is also reported.23 It is also concentration. Hence, the epicatechin and catechin fragments
speculated that M. smegmatis possesses multiple FabG, as both in our synthesized inhibitors play a crucial role in the inhi-
the organisms belong to the same family. Due to its fast bition potency. Compound 4 inhibited FabG4 with an IC50
growth and non-pathogenic nature, M. smegmatis was used in value of 213.3 µM (Fig. 8). However, the IC50 value of com-
this work instead of M. tuberculosis.
Both compounds have shown good anti-mycobacterial the inhibition potency clearly suggests the importance of the
properties. galloyl fragment linked to the 4-position of the triazole ring for
We used isoniazid (INH) as a reference compound for this the activity of the compound.
assay. The standardization of the assay conditions was carried These SAR findings are also supported by the docking
pound 1 is 44.7 µM. This approximately five-fold decrease of
out with different concentrations of inoculum and indicator studies. Compound 3 mainly floats over loop I of FabG4. The
using INH. The compounds were screened within the concen- 1,4-triazole ring only makes H-bonds with Asn295 at the pyro-
tration range of 0.5 μg mL−1 to 100 μg mL−1. The MICs of com- phosphate binding subsite (P-subsite); it shows no interactions
pounds 1 and 2 were found to be 20 μg mL−1 and 5 μg mL−1
whereas the MIC of INH was 4 μg mL−1 (see ESI†).
,
with the catalytic tetrad or NADH-binding amino acids. There-
fore, the overall docking score is poor. Compound 4 binds at
the major portal by making two hydrogen bonds with Asp244,
which is found to play a crucial role in NADH binding. It also
accesses the active site and weakly interacts with Tyr360
through the triazole ring, but it could not interact with the
nicotinamide-binding subsite or loop I due to the absence of
hydrogen bond acceptors/donors attached to the 4-position of
the triazole ring (see ESI†). In the future, we would like to
extend the SAR study by replacing the galloyl moiety with
other electron-rich aromatic moieties, and also examine the
role of the triazole ring by substituting with heterocyclic rings
such as imidazole and pyrimidine rings, which are capable of
forming H-bonds.
Structure–activity relationship (SAR)
The roles of fragments linked at the 1- and 4-positions of the
triazole rings in the inhibition activity have been evaluated by
this preliminary SAR study. For this purpose, fragments linked
to the 1- and 4-positions were substituted sequentially by
groups of less interacting sites, and then their activities were
compared with the original inhibitors. This strategy led to the
design of compounds 3 and 4. In compound 3, the catechin/
epicatechin fragment was substituted by a less interacting site,
a m-cresol unit. Similarly, in compound 4, the gallol fragment
linked to the 4-position of the triazole ring was replaced by a
benzyl unit,
a non-hydrogen bond forming counterpart
Conclusions
(Fig. 7). Here, compound 1 was taken as a model inhibitor to
compare the potential of compound 4.
We have successfully designed and synthesized inhibitors (tria-
zole polyphenol hybrid compounds) of the FabG4 enzyme of
Mtb for the first time. The compounds have shown good
inhibition at 0.88 μM and 3.97 μM respectively. Their design
strategy, synthesis, inhibition potential and properties are
discussed in this report. Their MIC values, as determined by a
resazurin assay against M. smegmatis, indicate their good anti-
mycobacterial properties. These compounds may be possible
candidates as lead compounds for alternate anti-tubercular
drugs. All of the reductase enzymes of the Mycobacterium
family have similar ketoacyl reductase (KAR) domains. Hence,
this work may be extrapolated to find structure-based inhibi-
tors of other reductase enzymes.
Experimental section
Docking details
The advanced and widely used molecular grid-based docking
program Autodock4.2 was used to predict the binding modes
and approximate binding free energies of all of the designed
inhibitors in the lead library. The X-ray crystal coordinates
of FabG4 were obtained from the Protein Data Bank
Fig. 7 Catechin/epicatechin fragment linked to the 1-position replaced
by a m-cresol unit, galloyl fragment linked at the 4-position of com-
pound 1 replaced by a less-interacting benzyl unit.
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Table 2 Anti-mycobacterial activities of the synthesized compounds
IC50 against
Mtb FabG4 (μM)
Ki against
Mtb FabG4 (μM)
MIC against
Compound
M. smegmatis (μg mL−1
)
44.7
3.97 0.02
20
34.9
0.88 0.01
5
Not found up to 400 μM
Not tested
Not tested
213.3
Not tested
Not tested
client. Energy minimization of the ligand structures was per-
formed by applying a CHARMM force field. The whole receptor
structure was selected for grid calculation with 0.375 Å spacing
to perform blind docking. Based upon the grid maps of all of
the atom types of the ligands, docking studies were carried out
by using the Lamarckian genetic algorithm. Standard proto-
cols were used, with an initial population of 150 randomly
placed individuals, a maximum of 2.5 × 106 energy evaluations,
a cluster tolerance of 2 Å (rms) and a maximum of 2.7 × 104
numbers of generations. After each docking execution, ten
resulting conformers were clustered according to their energy.
The conformer with the best binding energy was evaluated for
design purposes.
Chemistry details
Fig. 8 Dose–response plot to determine the IC50 value of compound 4
All of the reactions were conducted with oven-dried glassware
under an atmosphere of argon (Ar) or nitrogen (N2). All of the
common reagents were of commercial grade and used without
further purification. The solvents were dried by standard
methods and purified by distillation before use. Silica gel
(60–120 and 230–400 mesh) was used for column chromato-
graphy. TLC was performed on aluminum-backed plates
coated with Silica gel 60 with an F254 indicator. Locally avail-
able UV-lamp chambers and I2-blowers were used as the TLC
spot indicators. For solid compounds, melting points (MPs)
against FabG4.
(http://www.rcsb.org), PDB ID: 4FW8. The receptor structure
was edited and hydrogen atoms added prior to docking. Auto-
DockTools was used to assign Gasteiger charges to the recep-
tor. The catalytic tetrad and the other important amino acids
in the NADH binding region of the receptor were selected as
flexible residues to perform the flexible docking. The ligand
structures were built up using Accelrys Discovery Studio 3.1
80 | Org. Biomol. Chem., 2014, 12, 73–85
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were measured using melting point apparatus twice and
Yield: 600.5 mg (80%); state: white solid; mp: 90 °C; δH
reported without further calibration. The NMR spectra were (CDCl3, 400 MHz): 7.44–7.27 (15H, m, Ar–H), 6.67 (2H, s,
recorded on Bruker 200 MHz and 400 MHz machines. The fol- Ar–H), 5.10 (4H, s, 2 × –CH2Ph), 5.06 (2H, s, –CH2Ph), 4.56
lowing abbreviations are used to describe the peak patterns (2H, s, –CH2OH); δC (CDCl3, 50 MHz): 152.8, 137.7, 137.5,
where appropriate: s = singlet, d = doublet, t = triplet, q = 137.0, 136.6, 128.5, 128.4, 128.2, 128.1, 127.8, 127.7, 127.3,
quartet, m = multiplet, dd = double doublet, ABq = AB quartet. 106.2, 75.1, 65.2.
1,2,3-Tris(benzyloxy)-5-(bromomethyl)benzene
(12). Com-
pound 11 (511 mg, 1.2 mmol) was dissolved in dry CH2Cl2
(20 mL) and stirred in ice. Then PBr3 (1.5 eq.) solution in dry
General procedure for the synthesis of compounds 6 and 8
In solutions of compounds 5 and 7 (1 eq.) in dry THF, sodium DCM was added. The reaction mixture was stirred for 20 min,
hydride (4 eq.) was added at 0 °C and the mixture was stirred and was then carefully quenched with NaHCO3 solution and
for 30 minutes at 0 °C. Then propargyl bromide (2 eq.), dis- extracted with CH2Cl2 (3 × 30 mL). The CH2Cl2 layer was
solved in dry THF, was added dropwise. The mixture was washed with water 3–4 times, dried over sodium sulphate, fil-
stirred for 12 hours at room temperature, then quenched with tered and concentrated under vacuum. The pure compound
NH4Cl solution, and extracted with EtOAc. The organic layer was isolated as a gummy liquid by flash column chromato-
was washed with brine and dried over anhydrous sodium sul- graphy (Si-gel 60–120 mesh, PE–EA = 20 : 1 as the eluent).
phate. Evaporation under vacuum gave an oil, from which the
desired compounds were isolated by column chromatography 200 MHz): 7.40–7.25 (15H, m, Ar–H), 6.69 (2H, s, Ar–H), 5.09
(Si-gel 60–120 mesh, PE–EA = 7 : 1 as the eluent). (4H, s, 2 × CH2Ph), 5.03 (2H, s, CH2Ph), 4.39 (2H, s, CH2Br); δC
Yield: 499 mg (85%); state: yellow liquid; δH (CDCl3,
(2R,3R)-5,7-Bis(benzyloxy)-2-(3,4-bis(benzyloxy)phenyl)-3-(prop- (CDCl3, 50 MHz): 152.8, 137.7, 136.8, 133.1, 128.5, 128.1,
2-ynyloxy)chroman (6). Yield: 63%; state: white solid; mp: 127.9, 127.8, 127.4, 108.7, 75.2, 71.2, 34.2.
94 °C; δH (CDCl3, 400 MHz): 7.49–7.19 (21H, m, Ar–H),
5-(Azidomethyl)-1,2,3-tris(benzyloxy)benzene (13). 300 mg
7.01–6.91 (2H, m, Ar–H), 6.29 (2H, s, H-6 & H-8), 5.19 (4 h, m, of sodium azide (5 eq.) was added to 450 mg of compound 12
2 × CH2Ph), 5.04–4.97 (5H, m, H-2 & 2 × CH2Ph), 4.17 (1H, s, (0.92 mmol) in 30 mL dry DMF, and stirred at room tempera-
H-3), 3.99 (2H, m, OCH2), 3.04 (1H, m, H-4), 2.86 (1H, dd, J = ture for 12 h. Extraction was carried out with EtOAc (2 ×
17.2 Hz & 4 Hz), 2.24 (1H, broad s, –CCH).
25 mL), and the resultant solvent was washed by water, dried
(2R,3S)-5,7-Bis(benzyloxy)-2-(3,4-bis(benzyloxy)phenyl)-3-(prop- by sodium sulphate and subjected to column chromatography
2-ynyloxy)chroman (8). Yield: 60%; state: off-white solid; mp: (Si-gel 230–400 mesh, PE–EA = 15 : 1 as the eluent).
101 °C; δH (CDCl3, 400 MHz): 7.42–7.13 (20H, m, Ar–H), 7.00
Yield: 373.8 mg (90%); state: light yellow liquid; δH (CDCl3,
(1H, s, Ar–H), 6.87 (2H, broad s, Ar–H), 6.25 (1H, s, H-6/H-8), 200 MHz): 7.46–7.30 (15H, m, Ar–H), 6.66 (2H, s, Ar–H), 5.17
6.21 (1H, s, H-6/H-8), 5.17 (4H, m, 2 × CH2Ph), 4.99 (4H, m, 2 × (4H, s, 2 × CH2Ph), 5.11 (2H, s, CH2Ph), 4.28 (2H, s, –CH2N3);
CH2Ph), 4.80 (1H, d, J = 7.2 Hz, H-2), 3.93 (1H, m, H-3), 3.74 δC: 153.2, 137.9, 137.0, 131.1, 128.7, 128.3, 128.1, 128.0, 127.6,
(2H, m, OCH2), 2.97 (1H, dd, J = 16.4 Hz & 5.2 Hz, H-4), 2.68 108.0, 75.3, 71.4, 55.1.
(1H, dd, J = 16.4 Hz & 8 Hz, H-4), 2.32 (1H, broad s, CCH).
1-Methyl-3-(prop-2-ynyloxy)benzene (16). 500 mg m-cresol
Methyl 3,4,5-tris(benzyloxy)benzoate (10). 1.6 g of com- (9.3 mmol) was dissolved in 25 mL dry THF and cooled to
pound 9 (8.7 mmol) was dissolved in dry DMF (30 mL). 9.6 g 0 °C. 500 mg sodium hydride (4 eq.) was added under stirring
anhydrous K2CO3 (8 eq.) was added. 4.13 mL benzyl bromide and left for 20 min for anion generation. The reaction was
(4 eq.) was added portion-wise under stirring. After 24 h, the again cooled to 0 °C, and 1.1 mL of propargyl bromide (2 eq.)
reaction mixture was extracted with EtOAc (3 × 30 mL) and was added. Then the reaction was stirred at room temperature
washed with water. It was dried by sodium sulphate, concen- and monitored by TLC. After 48 h, the reaction was almost
trated and subjected to column chromatography (Si-gel completed. Then the excess sodium hydride was quenched by
60–120 mesh, PE–EA = 10 : 1 as the eluent).
adding saturated NH4Cl solution, extracted with EtOAc (3 ×
Yield: 2.9 g (75%); state: white solid; mp: 81 °C; δH (CDCl3, 40 mL) and purified through column chromatography (Si-gel
200 MHz): 7.45–7.26 (17H, m, Ar–H), 5.17 (6H, s, 3 × OCH2Ph), 60–120 mesh, PE–EA = 10 : 1 as the eluent).
3.92 (3H, s, CH3); δC (CDCl3, 50 MHz): 166.5, 152.5, 137.4,
136.6, 128.4, 128.1, 127.9, 127.9, 127.4, 125.1, 108.9, 75.0, 71.1, 200 MHz): 7.19 (1H, m, Ar–H), 6.81 (3H, m, Ar–H), 4.64 (2H, d,
52.1. J = 2.4 Hz, OCH2CCH), 2.49 (1H, t, J = 2.4 Hz, CCH), 2.35 (3H,
Yield: 1.1 g (78%); state: reddish liquid; δH (CDCl3,
(3,4,5-Tris(benzyloxy)phenyl)methanol (11). Compound 10 s, CH3); δC (CDCl3, 50 MHz): 157.7, 139.6, 129.3, 122.5, 115.9,
(800 mg, 1.76 mmol) was dissolved in dry THF (25 mL) and 111.8, 78.9, 75.5, 55.7, 21.6.
cooled to 0 °C. Lithium aluminium hydride (80 mg, 1.2 eq.)
General procedure for Huisgen’s 1,3-dipolar cycloaddition
was added very slowly under inert conditions. After 4 h, the
reaction mixture was diluted with EtOAc and an acid workup Compounds 14, 15, 17 and 19 were synthesized using this pro-
was carried out under cold conditions. The compound was cedure. The respective alkyne and azide counterparts were dis-
extracted with EtOAc (3 × 25 mL), washed with water and puri- solved in acetonitrile and degasified for 15 min. Copper
fied by column chromatography (Si-gel 60–120 mesh, PE–EA = sulphate (1 eq.) was dissolved in an equal volume of water and
7 : 1 as the eluent).
degassed for 30 min. Then sodium ascorbate (2 eq.) was
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added, and the solution was stirred for 15 min. Then the dd, J = 17.2 Hz & 3.6 Hz, H-4); δC (CDCl3, 100 MHz): 158.6,
degassed acetonitrile solution was slowly added. The mixture 158.0, 155.4, 148.5, 148.1, 145.6, 137.1, 137.1, 137.0, 136.9,
was stirred at room temperature until TLC indicated the dis- 134.6, 132.4, 128.9, 128.6, 128.5, 128.5, 128.5, 128.4, 128.4,
appearance of the starting materials. After that, the mixture 127.9, 127.8, 127.8, 127.8, 127.5, 127.4, 127.3, 127.2, 127.1,
was poured into saturated aqueous NH4Cl solution and the 122.5, 119.6, 114.5, 113.5, 101.3, 94.7, 93.8, 77.9, 72.1, 71.3,
product was extracted four times with EtOAc. The organic layer 70.9, 70.0, 69.8, 62.6, 53.7, 24.6. HRMS (ES+): calcd for
was dried with sodium sulphate and filtered, and the solvent C53H47N3O6 + H+ 822.3538, found 822.3494.
was removed under reduced pressure. The residue was purified
General procedure of debenzylation
by flash chromatography.
4-(((2R,3R)-5,7-Bis(benzyloxy)-2-(3,4-bis(benzyloxy)phenyl)- Debenzylation was carried out in a parr apparatus. Solutions
chroman-3-yloxy)methyl)-1-(3,4,5-tris(benzyloxy)benzyl)-1H- of compounds 14, 15, 17 and 19 in a mixture of THF–MeOH–
1,2,3-triazole (14). Yield: 70%, state: off-white solid; mp: H2O (20 : 1 : 0.5) were hydrogenated at 2–3 bar H2 pressure over
103 °C; δH (CDCl3, 400 MHz): 7.46–7.24 (35H, m, Ar–H), 7.06 20% Pd (OH)2–C for 5–7 h at room temperature. The reaction
(1H, s, Ar–H/olefinic H), 6.87 (2H, s, Ar–H), 6.48 (1H, s, Ar–H/ mixtures were filtered through celite using 1 : 1 ethyl acetate–
olefinic H), 6.41 (2H, s, Ar–H), 6.28 (2H, s, H-6 & H-8), methanol and concentrated in vacuo. The gummy liquid was
5.29–4.89 (17H, m, 7 × CH2Ph & NCH2 & H-2), 4.52 (2H, m, dissolved in a few drops of methanol and precipitation was
–OCH2), 3.99 (1H, broad s, H-3), 3.08 (1H, m, H-4), 2.83 (1H, carried out by adding diethyl ether. The compounds were puri-
dd, J = 17.2 Hz & 4 Hz, H-4); δC (CDCl3, 100 MHz): 158.7, 158.1, fied by repeated precipitation. The pure compounds appeared
155.4, 153.0, 148.7, 148.1, 145.5, 137.6, 137.1, 137.1, 137.0, as yellow gummy liquids in the absence of ether. All of the sol-
136.9, 136.7, 132.7, 130.2, 128.6, 128.6, 128.5, 128.4, 128.1, vents were distilled prior to use and inert conditions were
127.9, 127.9, 127.8, 127.5, 127.5, 127.4, 127.1, 122.5, 119.6, maintained for most of the time to avoid oxidation.
115.1, 113.4, 107.4, 101.4, 94.8, 93.5, 77.9, 75.1, 72.0, 71.7,
71.0, 71.0, 70.1, 69.9, 62.6, 53.8, 24.7; HRMS (ES+): calcd for 3-yloxy)methyl)-1H-1,2,3-triazol-1-yl)methyl)benzene-1,2,3-triol
C74H65N3O9 + H+ 1140.4794, found 1140.4791.
(1). Yield: 43%; state: yellow gummy liquid; δH (acetone-d6,
5-((4-(((2R,3R)-2-(3,4-Dihydroxyphenyl)-5,7-dihydroxychroman-
4-(((2R,3S)-5,7-Bis(benzyloxy)-2-(3,4-bis(benzyloxy)phenyl)- 400 MHz): 7.34 (1H, s, olefinic H, Ar–H), 7.11 (1H, s, olefinic
chroman-3-yloxy)methyl)-1-(3,4,5-tris(benzyloxy)benzyl)-1H- H, Ar–H), 6.88 (2H, d, J = 1.6 Hz, Ar–H), 6.46 (2H, d, J = 4.4 Hz,
1,2,3-triazole (15). Yield: 65%, state: off-white solid; mp: Ar–H), 6.11 (1H, d, J = 2 Hz, H-6/H-8), 6.00 (1H, d, J = 2 Hz,
107 °C; δH (CDCl3, 400 MHz): 7.41–7.23 (35H, m, Ar–H), 6.96 H-6/H-8), 5.33 (2H, ABq, J = 14.6 Hz, –NCH2), 5.02 (1H, s, H-2),
(1H, s, Ar–H/olefinic H), 6.88 (2H, s, Ar–H), 6.80 (1H, s, Ar–H/ 4.57 (2H, ABq, J = 12.8 Hz, –OCH2), 4.10 (1H, merged with
olefinic H), 6.46 (2H, s, Ar–H), 6.26 (2H, d, J = 2 Hz, H-6/H-8), ethyl acetate peak, H-3), 2.95 (1H, dd, J = 16.8 Hz, 3.2 Hz, H-4),
6.21 (1H, d, J = 2 Hz, H-6/H-8), 5.32–4.98 (16H, m, 7 × CH2Ph & 2.85 (1H, dd, J = 16.8 Hz, 4.2 Hz, H-4); δC (acetone-d6,
NCH2), 4.78 (1H, d, J = 7.6 Hz, H-2), 4.43 (2H, m, OCH2), 3.86 100 MHz): 156.7, 156.6, 156.3, 145.9, 145.2, 144.5, 144.4, 133.0,
(1H, m, H-3), 3.07 (1H, dd, J = 16.4 Hz & 5.2 Hz, H-4), 2.70 (1H, 131.3, 126.7, 122.7, 118.6, 114.6, 114.5, 107.3, 98.8, 95.4, 94.8,
dd, J = 16.4 Hz & 8.4 Hz, H-4); δC (CDCl3, 100 MHz): 158.7, 77.5, 73.4, 62.8, 53.3, 24.4; DEPT-135 (acetone-d6, 100 MHz):
157.6, 155.0, 153.0, 148.7, 148.5, 145.4, 138.4, 137.5, 137.0, 122.7, 118.5, 114.6, 114.4, 107.2, 95.3, 94.8, 77.4, 73.3, 62.7,
136.9, 136.8, 136.8, 136.6, 132.4, 130.0, 128.5, 128.5, 128.5, 53.3, 24.3. LCMS (ES−): calcd for C25H23N3O9 509.1434, found
128.4, 128.1, 127.9, 127.9, 127.8, 127.5, 127.4, 127.4, 127.3, 508.0723.
127.2, 122.4, 120.3, 114.9, 113.3, 107.5, 101.9, 94.1, 93.6, 79.5,
75.1, 74.5, 71.4, 71.0, 70.9, 70.0, 69.8, 62.9, 53.9, 25.3. HRMS 3-yloxy)methyl)-1H-1,2,3-triazol-1-yl)methyl)benzene-1,2,3-triol
(ES+): calcd for C74H65N3O9 + H+ 1140.4794, found 1140.4861.
(2). Yield: 40%; state: yellow gummy liquid; δH (acetone-d6,
5-((4-(((2R,3S)-2-(3,4-Dihydroxyphenyl)-5,7-dihydroxychroman-
4-(m-Tolyloxymethyl)-1-(3,4,5-tris(benzyloxy)benzyl)-1H-1,2,3- 400 MHz): 7.47 (1H, s, olefinic H), 6.86–6.80 (2H, m, Ar–H),
triazole (17). Yield: 75%; state: yellow floppy solid; mp: 6.69 (1H, m, Ar–H), 6.38 (2H, s, Ar–H), 6.01 (1H, d, J = 2 Hz,
132 °C; δH (CDCl3, 400 MHz): 7.34–7.16 (16H, m, Ar–H), 6.70 H-6/H-8), 5.88 (1H, d, J = 2.4 Hz, H-6/H-8), 5.29 (2H, ABq, J =
(3H, m, Ar–H), 6.44 (2H, s, Ar–H), 5.27 (2H, s, NCH2), 5.07 (2H, 14.4 Hz, –NCH2), 4.76 (1H, d, J = 7.2 Hz, H-2), 4.47 (2H, ABq,
s, OCH2), 4.96 (6H, s, 3 × CH2Ph), 2.23 (3H, s, CH3); δC (CDCl3, J = 12.4 Hz, –OCH2), 3.87 (1H, q, J = 7.2 Hz), 2.82 (1H, dd, J =
100 MHz): 158.1, 153.1, 144.6, 139.6, 138.5, 137.5, 136.6, 129.7, 16 Hz & 5.2 Hz, H-4), 2.55 (1H, dd, J = 16 Hz & 7.2 Hz, H-4); δC
129.2, 128.5, 128.5, 128.1, 127.9, 127.9, 127.4, 122.4, 122.0, (acetone-d6, 100 MHz): 157.8, 157.2, 156.7, 146.8, 145.7, 133.8,
115.5, 111.4, 107.7, 75.1, 71.1, 61.8, 54.2, 21.5. HRMS (ES+): 132.4, 127.8, 123.6, 119.6, 115.9, 115.0, 108.2, 108.1, 100.1,
calcd for C38H35N3O4 + H+ 598.2700, found 598.2696.
96.2, 95.4, 80.1, 75.5, 63.5, 54.2, 25.5; DEPT-135 (acetone-d6,
1-Benzyl-4-(((2R,3R)-5,7-bis(benzyloxy)-2-(3,4-bis(benzyloxy)- 100 MHz): 122.6, 118.6, 114.9, 114.0, 107.2, 95.2, 94.5, 79.1,
phenyl)chroman-3-yloxy)methyl)-1H-1,2,3-triazole (19). Yield: 74.5, 62.5, 53.2, 24.5; LCMS (ES−): calcd for C25H23N3O9
70%, state: white solid; mp: 97 °C; δH (CDCl3, 400 MHz): 509.1434, found 508.0962.
7.48–7.26 (23H, m, Ar–H), 7.17–7.11 (3H, m, Ar–H), 6.89 (2H, s,
5-((4-(m-Tolyloxymethyl)-1H-1,2,3-triazol-1-yl)methyl)benzene-
Ar–H), 6.59 (1H, s, olefinic H), 6.30 (2H, s, H-6 & H-8), 1,2,3-triol (3). Yield: 55%; state: deep yellow gummy liquid; δH
5.37–5.02 (10H, m, 4 × CH2Ph & NCH2), 4.90 (1H, s, H-2), 4.53 (acetone-d6, 400 MHz): 7.95 (1H, s, olefinic H), 7.13 (1H, t, J =
(2H, m, OCH2), 3.99 (1H, s, H-3), 3.08 (1H, m, H-4), 2.81 (1H, 7.8 Hz, Ar–H), 6.83–6.73 (3H, m, Ar–H), 6.41 (2H, s, Ar–H), 5.38
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(2H, s, –NCH2), 5.10 (2H, s, –OCH2), 2.27 (3H, s, –CH3); δC 25 °C. A reference power of 5 µcal s−1, stirring speed of
(acetone-d6, 100 MHz): 158.8, 146.0, 143.9, 139.3, 133.0, 129.2, 800 rpm and 120 s spacing were selected. The protein solution
126.9, 123.5, 121.7, 115.5, 111.7, 107.4, 61.5, 53.4, 20.7. HRMS was prepared in 50 mM HEPES buffer at pH 7.4. Protein solu-
(ES+): calcd for C17H17N3O4 + H+ 328.1292, found 328.1256.
tions of 10 μM, 4.3 μM and 3 μM concentrations were recorded
(2R,3R)-3-((1-Benzyl-1H-1,2,3-triazol-4-yl)methoxy)-2-(3,4- in the sample cell for the NADH (500 μM), compound 1
dihydroxyphenyl)chroman-5,7-diol (4). Yield: 53%; state: (1 mM) and compound 2 (1 mM) titrations respectively. The
yellow gummy liquid; δH (acetone-d6, 400 MHz): 7.35–7.29 (6H, ligand solutions were prepared in the same buffer with 3%
m, Ar–H), 7.04 (1H, s, olefinic H), 6.78 (2H, s, Ar–H), 6.01 (1H, HPLC grade methanol. This extra methanol was also added to
s, Ar–H), 5.89 (1H, s, Ar–H), 5.49 (2H, ABq, J = 14.8 Hz, the sample cell to avoid large heat changes due to solvent mis-
–NCH2), 4.94 (1H, s, H-2), 4.49 (2H, ABq, J = 12.8 Hz, –OCH2), match (Getting Started, MicroCal ITC200). Separate buffer–
4.01 (1H, s, H-3), 2.86–2.72 (2H, merged with water peak, 2 × ligand titrations were carried out as reference runs for each
H-4); δC (acetone-d6, 100 MHz): 156.8, 156.7, 156.4, 145.6, compound by recording the buffer in the sample cell. This
144.5, 136.2, 131.4, 128.9, 128.2, 128.1, 122.9, 118.7, 114.6, reference value was subtracted from the protein–ligand titra-
114.6, 98.9, 95.3, 94.8, 77.6, 73.3, 62.9, 53.3, 24.4. HRMS (ES+): tion to nullify the heat of dissolution. One injection of 0.4 μL,
calcd for C25H23N3O6 + H+ 462.1660, found 462.1671.
followed by nineteen injections of 2 μL of the ligand solutions,
were titrated into the FabG4 solution. All of the experiments
were repeated twice and the data were solved using MicroCal,
FabG4 inhibition assay
The inhibition studies were carried out in vitro at 25 °C by LLC ITC 200 software. All of the parameters, such as tolerance
monitoring the decrease of OD340 in the kinetic mode as a limit (zero), derivative delta (0.08) and weighting method (no
function of conversion of NADH to NAD+ using an Evolution™ weighting), were default values. The data points of the first
300 UV-visible spectrophotometer (Thermo Fisher scientific). injections (0.4 μL) were neglected. The heat changes of the 19
Protein purification was carried out as described elsewhere.24 injections (for the NADH and compound 1 titrations) and
Briefly, the recombinant protein was over-expressed in E. coli 18 injections (for the compound 2 titration) were plotted
M15 cells using IPTG induction. Cells were harvested and against the molar ratio of ligand.
lysed in a re-suspension buffer using ultra-sonication. The
lysate was centrifuged at 14 000 rpm for 40 min to discard cell
MIC study
debris. The supernatant was loaded onto a Ni-NTA column The Mycobacterium smegmatis mc2155 strain was grown in
and eluted with 300 mM imidazole solution. The eluted Middlebrook 7H9 broth with 0.2% glycerol and 0.05% Tween
protein was further purified by gel filtration chromatography 80 for 20 hours at 37 °C with shaking at 120 rpm, until the
using a Superdex200 column. The elutant was pooled and con- cells reached the mid-logarithmic phase (OD595 ∼ 0.5). The
centrated, and extensively dialyzed against HEPES buffer culture was diluted to OD595 = 0.0005 prior to addition into a
(50 mM, pH 7.4) containing 50 mM NaCl prior to the experi- sterile 96-well microtitre plate. Isoniazid (INH) was purchased
ments. The inhibitor compounds were dissolved in HEPES from Aldrich and used as a reference compound for this assay.
buffer (50 mM, pH 7.4) with 3% methanol (HPLC grade) to An INH stock solution of 1 mg mL−1 was prepared in distilled
prepare a stock solution of 1 mM, which was further diluted water and serially diluted in MB 7H9 media. 0.5 mg mL−1
repeatedly by HEPES buffer to achieve the desired concen- stock solutions of compound 1 and 2 (in 50 mM HEPES
trations. The reaction mixture contained the inhibitor solution buffer, pH 7.4, 3% HPLC grade methanol) were subjected to
in HEPES buffer, 0.5 mM acetoacetyl-CoA, 0.2 mM β-NADH serial two fold dilutions in MB 7H9 media to make the desired
and 1 μM of FabG4 protein in the final volume of 500 μL. A concentrations. Resazurin sodium salt was purchased from
mixture of FabG4, NADH and acetoacetyl CoA was used as a Sisco Research Laboratories, India. 0.5% (w/v) stock solution
positive control when no inhibitor was added. The reaction of resazurin was prepared in distilled water, filter-sterilized
was initiated by the addition of acetoacetyl-CoA. The decrease and diluted to 0.02% in distilled water.
in absorbance was recorded in 5 min intervals. The IC50 values
One hundred microlitres of diluted culture (OD595 = 0.0005)
were determined by varying the inhibitor concentrations until was added to each well of the microtitre plate. The total
full inhibition occurred. To determine the mode of inhibition, volume was two hundred microlitres. The inoculum was
we screened the enzyme activity with varying NADH concen- omitted from the negative control (Row A), and the inhibitors
tration at three different concentrations of inhibitor. All of the were omitted from the growth control (Row B). INH, the refer-
experiments were repeated thrice. The IC50 values were calcu- ence compound, was used as a positive control. After 24 hours
lated graphically from the dose–response plots. The modes of of post-drug incubation at 37 °C, 60 μL of 0.02% resazurin
inhibition were determined from Lineweaver–Burk plots. The solution was added to each well and incubated again for
inhibition constants were determined from secondary plots.
40 min at 37 °C. A color change from blue (resazurin) to pink
(resorufin) indicated the growth of bacteria. The lowest con-
centrations of drugs which prevented such a color change were
ITC experiments
Isothermal titration calorimetry (ITC) was carried out using a recorded as the MIC values. The assay was repeated twice and
Microcal ITC200 instrument. Titrations of FabG4 enzyme with an average of both the experiments was calculated to find the
compound 1, compound 2 and NADH were performed at MIC values.
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Acknowledgements
DRB thanks the Council of Scientific and Industrial Research
(CSIR) for the fellowship. Central Research Facility and Depart-
ment of Chemistry, IIT Kharagpur are thanked for providing
all of the instrumental facilities. AB and AKD gratefully
acknowledge the support of DBT for providing the necessary
research grant. AB is also grateful to DST for the JC Bose
National Fellowship. BS and KS are thankful to DST for fast-
track young scientist award.
10 P. Sharma, B. Kumar, N. Singhal, V. M. Katoch,
K. Venkatesan, D. S. Chauhan and D. Bisht, Indian J. Med.
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