Design, synthesis and characterization of dual inhibitors against new targets FabG4 and HtdX of Mycobacterium tuberculosis

Article abstract of DOI:10.1016/j.ejmech.2015.06.007

Herein, we present dual inhibitors of new targets FabG4 and HtdX for the first time. In this work, eight compounds have been designed, synthesized, characterized and evaluated for bio-activities. Amongst them, six compounds have shown inhibitory activities. Three of them (12e14) demonstrate dual inhibition of both FabG4 and HtdX at low micromolar concentration. In addition, the dual inhibitors show good anti-mycobacterial properties against both planktonic growth and biofilm culture of Mycobacterium species. This study is an important addition to tuberculosis drug discovery because it explores two new enzymes as drug targets and presents their dual inhibitors as good candidates for pre-clinical trials.

Full text of DOI:10.1016/j.ejmech.2015.06.007

Accepted Manuscript
Design, synthesis and characterization of dual inhibitors against new targets FabG4
and HtdX of Mycobacterium tuberculosis
Deb Ranjan Banerjee, Rupam Biswas, Amit K. Das, Amit Basak
PII:
S0223-5234(15)30082-9
10.1016/j.ejmech.2015.06.007
EJMECH 7934
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 1 February 2015
Revised Date: 21 May 2015
Accepted Date: 3 June 2015
Please cite this article as: D.R. Banerjee, R. Biswas, A.K. Das, A. Basak, Design, synthesis and
characterization of dual inhibitors against new targets FabG4 and HtdX of Mycobacterium tuberculosis,
European Journal of Medicinal Chemistry (2015), doi: 10.1016/j.ejmech.2015.06.007.
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ACCEPTED MANUSCRIPT
Design, synthesis and characterization of dual inhibitors against new targets
FabG4 and HtdX of Mycobacterium tuberculosis
Deb Ranjan Banerjee, Rupam Biswas, Baisakhee Saha, Amit K. Das* and Amit Basak*
Dual inhibitors 5a-5c of new targets FabG4 and HtdX of Mycobacterium tuberculosis are
reported.
=
==================================================================

ACCEPTED MANUSCRIPT
Design, synthesis and characterization of dual inhibitors against new
targets FabG4 and HtdX of Mycobacterium tuberculosis
a
b
b
+
a +
Deb Ranjan Banerjee , Rupam Biswas , Amit K. Das * and Amit Basak *
a
b
Department of Chemistry, Department of Biotechnology, Indian Institute of Technology,
Kharagpur 721302, India
AUTHOR INFORMATION
*
*
Corresponding Author
Phone +913222 283300. Fax +913222 282252
E-mail: absk@chem.iitkgp.ernet.in; amitk@hijli.iitkgp.ernet.in
ABSTRACT
Herein, we present dual inhibitors of new targets FabG4 and HtdX for the first time.
In this work, eight compounds have been designed, synthesized, characterized and evaluated
for bio-activities. Amongst them, six compounds have shown inhibitory activities. Three of
them (12-14) demonstrate dual inhibition of both FabG4 and HtdX at low micromolar
concentration. In addition, the dual inhibitors show good anti-mycobacterial properties
against both planktonic growth and biofilm culture of Mycobacterium species. This study is
an important addition to tuberculosis drug discovery because it explores two new enzymes as
drug targets and presents their dual inhibitors as good candidates for pre-clinical trials.
KEYWORDS
Tuberculosis, FabG4, HtdX, MIC, Biofilm
1

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INTRODUCTION
Tuberculosis (TB) still remains as one of the major infectious diseases worldwide;
approximately one third of global population is infected by its causative agent,
1
Mycobacterium tuberculosis (Mtb) and 1.3 million died from the disease in 2012. Due to
emergence of multi drug resistant (MDR) and extremely drug resistant (XDR) Mtb strains, a
numbers of first line TB drugs such as isoniazid, rifampicin, pyraniazid, ethambutol and
2
streptomycin have failed in recent TB cases. Besides, treatment of tuberculosis is a long-
time procedure that requires almost 6-9 months of multiple antibiotic therapies to avoid the
3
re-emergence of the disease. The reason behind this long treatment procedure is biofilm-like
4
pellicle growth by Mtb which is distinct from planktonic growth. The pellicle shows
increased phenotypic resistance and harbors drug-resistant Mtb cells which persist despite
exposure of high concentrations of antibiotics. In this persistent stage, Mtb remains in slow
growing or non-growing state and leads to the asymptomatic latent infection. Thus, the
combined effects of both genetic and phenotypic resistance by Mtb is forcing the scientific
community to come up with new drug targets and alternate drugs which are crucial to stop
this ancient pathogen from further killing.
Mtb possesses lipid rich cell wall, produced by fatty acid synthesis (FAS) pathway,
required for its survival within host cell. Mtb cells become virtually impenetrable to the
antibiotics due to presence of the thick lipid rich envelop. FAS pathway not only required in
5
planktonic growth but also implicated in the formation of pellicle during the latent infection.
6
The FAS generally consists of two different pathways, namely FAS-I and FAS-II. FAS-I is a
multi-domain enzyme, involved in the de novo synthesis of short chain fatty acids. FAS-II is
involved in the production of very long chain fatty acids and composed of discrete mono-
functional enzymes. Apart from FAS I and FAS II, many bypass fatty acid biosynthetic
pathways are possibly present in Mtb and these have been implicated to its drug resistance
7
, 8, 9
behavior.
Besides, the inherent redundancy of polygenic Mtb network makes it
impossible to shut down a cellular pathway by taking out a single target. Thus, the enzymes
involved in bypass pathways provide attractive targets for alternate anti-TB drug discovery.
Our targets FabG4 (Rv0242c) and HtdX (Rv0241c) are two such less explored enzymes that
belong to a single fatty acid metabolizing operon of Mtb (Figure 1). This operon is conserved
among most actinobacteria and some lipid rich proteobacteria and might be involved in
8
10
interlinked CoA dependent fatty acid metabolism pathway instead of traditional FAS
pathway. The FabG4, a β-ketoacyl CoA reductase, is recently reported to be an essential and
2

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1
1
12
functional protein for bacterial growth, survival and fatty acid synthesis. There are five
1
3
fabG genes present in Mtb genome, but only fabG1 (rv1483) and fabG4 (rv0242c) are
conserved among mycobacterial species. FabG4 is possibly involved in genetic resistance of
1
4
Mtb as it is over-expressed in sub-inhibitory concentrations of streptomycin. It is also linked
1
5
with pellicle formation as it is over-expressed in biofilm mode of growth. Thus, it is a
potential target for alternative drugs that can be administered with frontline TB drugs to
shorten the TB treatment time. The other enzyme of interest is HtdX (Rv0241c), a 3(R)-
hydroxyacyl CoA dehydratase, is also a member of the conserved operon. The enzyme
functions in a dehydration step after reduction of ketoacyl substrate by FabG4 (Figure 1).
Alike FabG4, HtdX is also a physiologically functional and essential for the bacillus growth
1
6
and fatty acid production. The enzyme is hypothesized to be involved in bypass fatty acid
8
, 10c, 17, 18
synthesis pathway and in drug resistance of Mtb.
There are eleven putative (R)-
specific hydratase/dehydratase candidates identified in Mtb, but only HadB (Rv0636) and
HtdX are - (i) ubiquitous among every mycobacteria and related mycolate-producing genera;
1
9
(ii) contain characteristic and well conserved catalytic sequence, called ‘hydratase 2 motif’.
In vitro activity of purified HtdX has been checked and it has shown 3-hydroxyacyl
dehydratase/trans-2-enoyl hydratase activity with preference for CoA as carrier unit of the
1
0c
acyl chain . Also, HtdX and FabG4 can plausibly function within a shared pathway as they
1
6, 18, 20
both belong to single operon.
This type of pair arrangement has been observed in other
6
enzymes of FAS family, including FabG1 (Rv1483) and InhA (Rv1484). These reports and
facts have made HtdX a potential target for the drug discovery along with FabG4. Recently,
2
0
18
the crystal structures of FabG4 and HtdX have been solved which build up the platform to
design structure-based inhibitors against the enzymes concerned.
As FabG4 and HtdX are involved in consecutive steps of fatty acid metabolism and
plausibly function within a shared interlinked pathway, a single inhibitor that can inhibit both
FabG4 and HtdX may result in synergistic effect in overall therapeutic efficacy. Moreover, a
2
1
single drug that can simultaneously interact with multiple targets (polypharmacology) is
2
2
found to be more efficient against advance-stage polygenic pathologies compared to a
combination drugs with high specificity for a different targets. Combination of drugs
(combination therapy) has several drawbacks like drug-drug interaction, increased toxicities
2
3
compared to a single drug with multi-target efficiencies. Additionally, multi-target single
therapeutic agent has more predictable pharmacokinetic and pharmacodynamic properties
2
3
and less adverse effect due to administration of a single drug. Inspired by these prospects,
3

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we undertook the task to develop dual inhibitors of both FabG4 and HtdX for the first time.
The strategy to design dual inhibitors through blending of structure-based and ligand-based
design is described. These designed hybrids have been synthesized, characterized and
evaluated for their bio-activities. Inhibition profiles have been analyzed by various
biophysical methods (inhibition kinetics, ITC, CD) and molecular docking study. In addition,
growth inhibition assays of these novel dual inhibitors has been checked against both
planktonic and biofilm growth of Mycobacterium.
RESULTS AND DISCUSSION
DESIGN CONCEPT FOR DUAL INHIBITOR
Rational design of multi-targeting inhibitor remained as a challenging task for a
medicinal chemist, particularly for a combination of structurally divergent targets, namely
FabG4 and HtdX for the present case. A good design strategy determines the difference
2
3
between ‘targeted polypharmacology’ and ‘drug promisquity’. Considering this scenario,
the number of articles containing rational designing of drugs with desired multi-target
profiles has increased steadily in recent times. One such design strategy adopts combination
2
4
of common pharmacophore selection and molecular docking study. For our case, we adopt a
-steps design strategy that combines both structure-based and ligand-based designing.
3
Step 1: Target site analysis of FabG4 and HtdX
The crystal structure of FabG4 with co-factor NADH and substrate Hexanoyl CoA
(PDB ID: 3V1U) shows that the active site of FabG4 could be accessed from two different
sides: a narrow minor portal and a wide major portal. The co-factor NADH binds at the
relatively wide major portal, while thinner fatty acyl substrate accesses the active site via
2
5
minor portal (Figure 2). For NAD-dependent enzyme like FabG4 where substrate binding
site (minor portal) is narrow and intrinsically non-druggable, NAD binding pocket (major
2
6
portal) can be a viable drug target. The NAD binding site can be divided into three subsites,
the nicotinamide binding subsite (N-subsite), the adenosine binding subsite (A-subsite) and
the pyrophosphate binding subsite (P-subsite). If a ligand binds at all or any of these subsites,
it will selectively repel NAD and thus should act as an inhibitor of the enzyme. Based on
these structural insights, we targeted NAD binding pocket for inhibitor designing against
FabG4. In addition, the active site of FabG4 is composed catalytic tetrad Ser347, Tyr360,
Lys364 and Asn319. The active site is covered by two catalytic loops, namely loop-I and
4

ACCEPTED MANUSCRIPT
loop-II. Movement of these catalytic loops is crucial during the enzymatic action of FabG4.
Loop I consists of a conserved NAG triad (Asn295, Ala296, Gly297) which interacts with
pyrophosphate part of the NADH and directs the cofactor towards active site.
The crystal structure of HtdX (PDB ID: 3WEW) shows that it has a characteristic
1
8
double hot dog fold (DHD) motif. Alignment of amino acid sequence of HtdX with M.
tuberculosis HadB, eukaryotic peroxisomal dehydrogenase and hydratase 2 has shown that
the catalytic loop of HtdX consists of 26 amino acids (Gly161-Gly185) (Figure 3). In this
catalytic loop, active site residues Asp162 and His167 provide the conserved basic hydratase
1
9
2
motif consisting of [Asp]-x(4)-His] sequence. Couple of glycines at both terminals of the
catalytic loop governs the flexibility for the binding of variable hydrophobic tails of
substrates. In addition, the active site cavity is covered by hydrophobic lid comprising Ile171,
Leu175, Phe176 and Phe178 (Figure 3). The inner cavity of catalytic tunnel is negatively
charged and acidic. Therefore, a ligand can inhibit the HtdX by occupying the hydrophobic
entrance and plugging the hydrophilic catalytic tunnel via interacting with the active site
residues. Based on these structural insights, we targeted catalytic loop of HtdX for inhibitor
designing.
Step 2: Selection of common pharmacophores
Based on structural knowledge of the target sites, we chose pharmacophores from
ligand-based screening. For the enzymatic action of both FabG4 and HtdX, movements of the
loops (loop I-loop II of FabG4 and catalytic loop of HtdX) are essential. To restrict the loop
movements, we chose conformationally constrained β-lactam moiety as a common
pharmacophore in our design. Besides, the carbonyl group at C-2 of β-lactam ring can
interact with the protein residues and delivers the rigidity to the binding site. Isoniazid (INH)
was chosen as another pharmacophore based on following rationale: Isoniazid is a bio-
isostere of nicotinamide ring due to structural and chemical similarity between the two.
Hence, Isoniazid is a potential N-subsite binder of NADH binding site and a natural
pharmacophore of FabG4. Again, HtdX catalytic tunnel is negatively charged and acidic;
while Isoniazid is basic and partially positively charged. Hence, Isoniazid is a potential
binder at the catalytic tunnel of HtdX due to favourable charge interaction. Besides, Zhang et
2
7
al have reported nicotinic hydrazide (isomer of isoniazid) linked Schiff bases as competitive
inhibitor of another dehydratase enzyme (HpFabZ) involved in bacterial FAS II. In addition,
5

ACCEPTED MANUSCRIPT
aromatic rings were also chosen as common pharmacophores in order to interact with the
hydrophobic lid of HtdX catalytic loop.
Step 3: In silico selection
After selecting the common pharmacophores for both the targets, we merged them to
obtain a small library. From the library, final candidates were selected using stepwise
docking studies. Following this procedure, we designed seven compounds (8-14, Figure 4) as
candidates for later parts of this work. Amongst them, compounds 12-14, that contain
Isoniazid as a fragment of their structure, showed most promising docking results against
both FabG4 and HtdX. Additionally, compound 16 was designed through replacing Isoniazid
fragment of compound 12 by non-polar benzhydrazide fragment.
CHEMISTRY
The methodology to obtain the designed compounds is shown in Scheme 1 and 2. The
2
8
key step to obtain the designed compounds was Kinugasa reaction involving cycloaddition
between various nitrones and in situ generated copper acetylide from ethyl propiolate which
provided the β-lactam core. In this synthesis procedure, no protecting group was necessary
that reduced the number of steps and increased the overall yield.
The various nitrones employed as the partner for Kinugasa reaction were prepared
starting from the phenyl hydroxylamine which was reacted with various aldehydes (4-
pyridinecarboxaldehyde/2-thiophenecarboxaldehyde/2-furan
carboxaldehyde)
in
dry
methanol that led to the desired nitrones (2,3,4). The Kinugasa reactions were performed in
between the respective nitrone and ethyl propiolate in presence of CuI (1 eq) in dry DMF to
obtain β-lactam esters (5,6,7). The esters were hydrolysed carefully at low temperature using
0
.1N LiOH in methanol-water (1:1) solvent to obtain corresponding β-lactam acids (8,9,10)
(Scheme 1). Additionally, compound 11 was synthesized via reduction of compound 8 with
sodium borohydride in methanol.
Subsequent treatment of β-lactam acids produced rest of deigned compounds as
depicted in Scheme 2. Isoniazid linked compounds (12-14) were synthesized by EDC-
mediated coupling of β-lactam acids (8-10) with Isoniazid. For the synthesis of compound 16,
benzhydrazide (15) was prepared from benzoic acid via chlorination followed by coupling
with hydrazine hydrate. Subsequent EDC-mediated coupling with β-lactam acid 8 furnished
compound 16.
6

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1
13
All final compounds were characterized by H, C NMR and mass spectra. Purity of
these compounds was checked through reverse-phase analytical HPLC (selective traces are
given in SI). Finally, structure of one Isoniazid linked derivative was confirmed through X-
ray crystal structure determination of 14 (ORTEP shown in Figure 5).
INHIBITION KINETICS
Inhibition of FabG4
Inhibition kinetics of compounds 8-14 and 16 was carried out against FabG4 at 25 °C
by monitoring the decrease in absorbance at 340 nm due to the conversion of NADH to
+
NAD . Half maximal inhibitory concentration (IC ) values were evaluated by varying
5
0
inhibitor concentrations until full inhibition occurred. Amongst the β-lactam acids (8,9,10),
only compound 8 that contains pyridyl substituent at C-4 of β-lactam ring, inhibits the
enzyme at micromolar concentration (IC =64.3±1µM). This result shows that pyridyl
5
0
substituent has significant influence on FabG4 inhibition as compared to thiophenyl/furyl
substituent and corroborates with the fact that NADH binding pocket is a good target of
2
9
pyridine nucleus containing natural product like pyridomycin. Moreover, NADH is itself a
highly polar compound and its binding pocket naturally prefers polar substituent like pyridine
ring than hydrophobic substituent like thiophene or furan ring. Docking study of compound 8
with FabG4 indicates that pyridine ring binds near the N-subsite, whereas the adjacent
carboxyl acid group interacts with nearby active site residue. Inhibition kinetics of compound
1
1 that contains pyridyl substituent at same position was used to crosscheck the finding.
Compound 11 also inhibits FabG4, but the inhibitory power is minimum
IC =666.1±20µM); that may be due to the replacement of carboxylic acid group of 8 by
(
5
0
alcohol moiety adjacent to pyridyl ring.
All Isoniazid linked hybrids (12,13,14) with most promising docking results inhibited
FabG4 at low micro molar concentrations. Amongst them, hybrid 12 that contains pyridine
substituent attached to β-lactam ring showed highest inhibition (IC =15.2±0.5µM) of FabG4
5
0
enzyme. Although, thiophene and furan containing β-lactam acids (9,10) could not inhibit
FabG4 up to 1mM concentration, but their isoniazid coupled products (13, 14) inhibited
FabG4 enzyme at low micromolar concentrations. Above result validates our designing
strategy that Isoniazid fragment plays a crucial role in binding of these compounds at NADH
binding pocket. Moreover, the benzhydrazide linked compound (16) exhibited approximately
three times less inhibitory potency (IC =39.9±2µM) than its Isoniazid bound analogue (12)
5
0
7

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and that further elucidates the practical implication of Isoniazid in FabG4 inhibition. Mode of
inhibition of the best found inhibitor (12) was evaluated against FabG4 with varying NADH
concentration at three different concentrations of inhibitor. The compound 12 was found to be
competitive with respect to NADH. Competitive inhibition also substantiates with designing
that these inhibitors target NADH binding site.
Inhibition of HtdX
The enzyme assay of HtdX was performed spectrophotometrically (at 263 nM) in
1
0c
presence of crotonoyl-CoA as substrate. Inhibition kinetics of compounds 8-14 and 16
were carried out to screen their inhibition potencies. The β-lactam acids (8,9,10) and
compound 11 were found to be inactive against HtdX up to 1mM concentration. But
Isoniazid linked compounds (12,13,14) demonstrated strong HtdX inhibition at low
micromolar concentrations. The hybrid 13 with aromatic thiophene substituent at C-4 of β-
lactam ring showed best inhibition (IC =10.3±0.8µM) of HtdX; whereas the hybrid
5
0
compound 12 with polar pyridine substituent at the same end showed somewhat less
inhibition (IC =22.3±0.5µM). This inhibitory tendencies verifies our design strategy that
5
0
aromatic substituent linked to β-lactam ring assist the inhibitor to bind at catalytic loop via
hydrophobic aromatic interaction with the hydrophobic lid at the entrance of the loop. The
benzhydrazide linked compound (16), the analogue of compound 12 with benzhydrazide
fragment in place of isoniazid fragment, could not inhibit HtdX up to 1mM concentration.
The inability of compound 16 to inhibit HtdX strongly demonstrates the significance of
isoniazid fragment in HtdX inhibition and indicates polar interactions with the isoniazid
fragment with the hydrophilic catalytic tunnel residues is the main driving force behind the
inhibitor binding. Additionally, mode of inhibition of the best found inhibitor (13) was
checked with varying crotonoyl-CoA concentration at three different concentrations of
inhibitor. Compound 13 is found to be competitive with respect to crotonoyl-CoA. This
competitive mode of inhibition supports our claim that the designed inhibitors bind at the
catalytic loop and prevent the fatty acyl substrate to enter in to the catalytic tunnel.
Dual inhibition of FabG4 and HtdX
From the listed in vitro inhibition profiles of all designed and synthesized compounds
against FabG4 and HtdX (Table 1), it can be clearly seen that this study successfully leads to
the identification of three novel compounds (12,13,14) as potent dual inhibitors of FabG4 and
8

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HtdX with IC values in low micromolar range. Amongst them, compound 12 showed best
5
0
inhibition of FabG4; while compound 13 was found as best inhibitor of HtdX.
ISOTHERMAL TITRATION CALORIMETRY (ITC)
FabG4 titration
Isothermal titration calorimetry (ITC) of FabG4 with the best found inhibitor
(compound 12) was performed at 25 °C to obtain the thermodynamic parameters of binding.
To understand the binding nature of compound 12, FabG4-12 titration was compared with
FabG4-NADH titration (as NADH was used as a substrate in inhibition kinetics). FabG4
exists as an inseparable homo-dimer in solution and two NADH molecules bind sequentially
3
0
to each monomer with negative co-operativity. Thus, NADH binding to first monomer is
co-operative to NADH binding to second monomer and the co-operativity effect carries
3
1
through dimeric interface. The binding curve of FabG4-12 titration fits well to the
sequential binding’ mode with number of binding site (N = 2). Bindings of compound 12
with both the monomers of FabG4 dimer are spontaneous as indicated by negative ꢀG values
Table 2). Moreover, negative ꢀH and ꢀS values signify enthalpically favoured bindings and
interactions via good hydrogen bonding and conformational changes. The first binding
‘
(
5
-1
3
-1
constant (K = 2.98 x 10 M ) is higher than second binding constant (K = 1.15 x 10 M )
1
2
which indicates negative co-operativity. Thus, calorimetry of compound 12 with FabG4
reveals that it also binds sequentially to each monomer of FabG4 dimer with negative co-
operativity as followed in case of NADH. Therefore, this similarity in binding nature of
FabG4-NADH binding and FabG4-12 binding with a distinct trend of negative cooperativity
at two sites of FabG4 dimer supports that designed inhibitors binds at the NADH binding
pocket of FabG4.
HtdX titration
ITC was used to evaluate the parameters of binding between HtdX and its best found
inhibitor (compound 13). HtdX-13 titration was carried out at 25 °C. The integrated binding
isotherm fits to the ‘one site’ binding model with number of binding site (N) close to one.
The binding of compound 13 with HtdX is spontaneous (negative ꢀG) with high binding
5
-1
constant ((K = 4.21 x 10 M ) (Table 2). The overall binding process is enthalpically
favoured (negative ꢀH) that indicates the polar interaction dominates in binding. The
9

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negative ꢀH along with negative ꢀS value indicates interactions via hydrogen bonding and
conformational changes which support our design concept.
CIRCULAR DICHROISM (CD)
Circular dichroism (CD) was conducted to evaluate the change in secondary
structures of proteins on binding of these dual inhibitors. For this, CD studies were carried
out between FabG4 and 12, and HtdX and 13 in Jasco-815 automatic spectropolarimeter at 25
ºC. In both cases, the molar concentration ratio was 1:5 (protein:ligand) in the micromolar
range. Ligands were taken in reaction buffer (Tris) and used for baseline correction followed
by two separate runs; one is only protein and another is protein-ligand mixture. The CD
spectra of free proteins and their complexes are shown in supporting information. The
changes in secondary structures of FabG4 and HtdX on interaction with 12 and 13 were
analyzed and given in Table 3.
The percentage of α-helix of FabG4 increases on interaction with 12. The helicity
3
2
enhancement is related with co-operativity and the change in secondary structure supports
our previous finding from ITC that FabG4-12 binding is cooperative in nature. In case of
HtdX, helicity and turns slightly decrease, and percentage of beta sheet increases on
interaction with 13.
MOLECULAR DOCKING STUDY
Molecular docking studies were carried out as a part of design strategy. Here, docking
results of FabG4-12 and HtdX-13 are described in details. Results of additional docking
studies are included in supporting information
Isoniazid linked compound 12, best found inhibitor against FabG4, binds at the major
portal of FabG4 and blocks all three subsites (A-, P-, N-) of NADH binding site. Moreover,
the Isoniazid fragment is directed towards the N-subsite and interacts with catalytic residues
Tyr360 via hydrogen bonding interaction and with Lys364 via cation-π interaction. The β-
lactam ring along with bis-amide linker binds loop I (P-subsite) and interacts with crucial
Gly297 residue via hydrogen bonding interactions. The A-subsite of NADH binding site has
been occupied by the aromatic substituent linked to β-lactam ring. In between them, the
pyridine substituent makes hydrogen bond with Asp244 and the phenyl substituent
experiences a sigma-π type interaction with Ala296 of loop I (Figure 6A).
1
0

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Isoniazid linked compound 13, the best found inhibitor against HtdX, binds at the
catalytic loop of HtdX. The aromatic substituent linked to β-lactam ring interacts with the
hydrophobic outer lid; while the isoniazid fragment directs towards the inner side of catalytic
tunnel. The phenyl and thiophene substituents at 1- and 4- position of lactam ring interact
with Phe178 via hydrophobic aromatic interactions. The carboxy goup at 3-position of lactam
ring makes hydrogen bond with terminal Gly185, a crucial residue for the flexibility and right
conformation of the catalytic loop. Finally, the isoniazid fragment interacts with catalytic
residue His167 via hydrogen bond formation and with Asp162 via charge interactions
(Figure 6B).
MINIMUM INHIBITORY CONCENTRATION (MIC ASSAY)
Anti-mycobacterial activity of the found dual inhibitors was evaluated against
3
3
Mycobacterium smegmatis using aerobic resazurin microplate reduction assay (REMA).
The M. smegmatis is non-pathogenic, fast growing and contains both fabG4 and htdX genes
1
5, 19
in its genomic sequence with high degree of conservations.
For these reasons, M.
2
smegmatis mc 155 strain was chosen as a model organism for this study. The assay
conditions were standardized using different concentrations of inoculum, resazurin indicator
and reference compound isoniazid (INH). Concentrations of dual inhibitor 12, 13 and 14
-1
-1
were screened in the range of 0 µg mL to 100 µg mL . Dual inhibitors of FabG4 and HtdX
-1
-
has shown good anti-mycobacterial activities with MIC value 15 µg mL (for 12), 20 µg mL
1
-1
(
for 13) and 15 µg mL (for 14), whereas the MIC of reference compound Isoniazid is
-1
found to be 10 µg mL (REMA plate image is included in supporting information).
It may be mentioned that MIC of synthesized compounds is probably not due to free
Isoniazid liberated through breaking of amide bond (at C-3 of β-lactam) in cellular condition.
Because, amide bond at C-3 of β-lactam ring is generally considered as stable in cellular
condition as many commercial β-lactam antibiotics (such as Penicillin G, Amoxicillin etc.)
contain amide linker at same position in their structures. Amongst them, Amoxicillin is
34
reported to be functional inside mycobacterial cell. Also, the monocyclic β-lactam rings are
3
5
more stable than bicyclic β-lactam rings and they are least prone to nucleophilic ring
opening when no electron withdrawing group is attached to N-1. To crosscheck this
possibility, we separately incubated the dual inhibitor 12 in culture media in presence of M.
smegmatis for 24 h afterwhich, the culture was lyophilized, dissolved in solvent and
subjected to HPLC. No trace of Isoniazid was seen in HPLC (traces are included in SI).
1
1

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Above data supports that anti-mycobacterial activity of synthesized dual inhibitors is due to
the activity of intact compound and not due to any release of isoniazid during incubation. .
BIOFILM INHIBITION ASSAY
As already mentioned FabG4 is over-expressed during biofilm formation and linked
to latent infection. It is also probable that FabG4 and HtdX, members of same operon,
function through interlinked pathway. Hence, we have screened the synthesized dual
inhibitors of FabG4-HtdX pair against biofilm formation by Mycobacterium species. Again,
non-pathogenic M. smegmatis has been chosen as model organism as it contains both fabg4
1
5, 19
and htdX genes in its genome sequence.
Besides, biofilm formation in M. smegmatis is
Compound 12 was chosen for this study and its MIC against biofilm
formation was determined. It showed complete inhibition of biofilm formation at
4
,5,36
well characterized.
-1
concentration of 20 µg mL (plate image is included in SI). The result is interesting in view
3
6
of reported resistance of M. smegmatis biofilm culture against Isoniazid
CONCLUSION
This is the first report of dual inhibitors against FabG4 and HtdX. Possible
involvements of FabG4 and HtdX in bypass fatty acid synthesis pathway, drug resistance and
latent infection have implicated them as alternative targets in anti-tuberculosis research due to
the unavailability of drugs that can act against drug resistant Mtb strain or latent stage
tuberculosis. Hence, the dual inhibitor of FabG4 and HtdX has immense potential to be used
in pre-clinical trials In addition, polypharmacology of these dual inhibitors may also results in
therapeutic synergy in vivo as both FabG4 and HtdX enzymes belong to single operon and
involve in successive steps of fatty acid metabolizing procedure. The dual inhibitors have
been designed, synthesized, characterized and evaluated for bio-activities. These hybrids
inhibit FabG4 and HtdX at low micromolar concentration and show good anti-mycobacterial
property against both planktonic growth and biofilm culture. Thus, this study successfully
explores FabG4 and HtdX as new anti-TB targets and presents their dual inhibitors as
possible lead compounds against the resistant/dormant mycobacterial strain.
EXPERIMENTAL SECTION
Molecular docking
1
2

ACCEPTED MANUSCRIPT
Advanced and widely used molecular grid-based docking program Autodock4.2 was
used for molecular docking studies. The X-ray crystal coordinates of FabG4 (PDB ID 4FW8)
and HtdX (PDB ID 3WEW) were obtained from the Protein Data Bank
(http://www.rcsb.org). Prior to dockings, receptor structures were edited by adding hydrogen
atoms and gasteiger charges in autodocktools to create PDBQT files. Crucial residues at
probable binding sites of receptors were selected as flexible residues to perform flexible
dockings. Ligand PDB structures were built up using Accelrys Discovery studio 3.1 client.
Energy minimization of ligand structures were carried out using CHARMm force field.
Ligand PDB files were edited in autodocktools by defining root center, aromatic carbons,
torsions and saved as PDBQT files. In grid selections, the whole receptor structures were
selected for autogrid calculation with 0.375 Å spacing to perform blind dockings. Finally,
docking studies were carried out using Lamarckian genetic algorithm based on the grid maps
of all atoms present in the receptors and ligands. Parameters were used with an initial
7
population of 150 randomly placed individuals, a maximum of 2.5x10 energy evaluations,
4
cluster tolerance of 2 Å (rms), output level 1 and maximum of 2.7x10 numbers of
generations. After each docking execution, the docked conformers were analyzed by
autodocktools, pymol and discovery studio 3.1 client. The images were made using pymol
viewing software.
Chemical synthesis and analysis
All common reagents were commercial grade reagents and used without further
purification unless necessary. All reactions were conducted with oven-dried glassware under
an atmosphere of argon (Ar) or nitrogen (N ).The solvents were dried by standard methods
2
and purified by distillation before use. Silica gel (60–120 and 230–400 mesh) and alumina
(neutral and basic) were used for column chromatography. TLC was performed on
aluminum-backed plates and UV-lamp chamber or I -blowerwas used as the TLC spot
2
indicator. The NMR spectra were recorded at Bruker 200 MHz and 400 MHz machine. The
following abbreviations are used to describe peak patterns where appropriate: s = singlet, d =
doublet, t = triplet, q = quartet, m = multiplet, dd = double doublet, ABq = AB quartet.
General procedure for the synthesis of nitrones (2,3,4)
1
3

ACCEPTED MANUSCRIPT
A solution of the aldehyde (4-pyridine carboxaldehyde/thiophen-2-aldehyde/furfural,
0 mmol) and phenyl hydroxyl amine (10 mmol) in dry methanol (30 mL) was stirred in
1
room temperature for 12 h under argon atmosphere. Upon completion of the reaction as
indicated by TLC, the solution was dried under vacuo to leave a residue from which pure
nitrones were isolated by flash column chomatography (Neutral alumina for 2, Si-gel for 3, 4)
using 2:1 PE/EA as eluent. Spectral characteristics of the purified compounds are mentioned
below:
Compound 2: Yield: 80%; State: light yellow solid; mp: 145 °C ; δ (CDCl3, 200 MHz):
H
8
.75 (2H, d, J = 7.1 Hz), 8.17 (2H, d, J = 5.4 Hz), 7.96 (1H, s), 7.74-7.79 (2H, m), 7.49-7.53
(
3H, m); δ (CDCl3, 50 MHz):150.5, 136.9, 132.4, 132.4, 130.7, 129.3, 121.7, 121.6.
C
Compound 3: Yield: 85%; State: light yellow solid; mp: 75 °C; δ (CDCl3, 200 MHz): 8.48
H
(
1H, s), 7.75-7.80 (2H, m), 7.37-7.58 (5H, m), 7.14 (1H, m); δ (CDCl3, 50 MHz):146.3,
C
1
32.9, 131.2, 130.0, 129.8, 129.1, 129.0, 127.0, 120.9.
Compound 4: Yield: 89%; State: light yellow solid; mp: 78 °C; δ (CDCl3, 400 MHz): 8.17
H
(1H, s), 8.02 (1H, d, J = 3.6 Hz), 7.60-7.80 (2H, m), 7.59 (1H, s), 7.45-7.51 (3H, m), 6.65-
6
1
.66 (1H, m); δ (CDCl3, 50 MHz):147.6, 144.9, 130.1, 129.4, 124.7, 121.2, 116.9, 112.9,
C
12.7.
General procedure of Kinugasa reaction for the synthesis of β-lactams (5,6,7)
To degassified dry DMF (20 mL), ethyl propiolate (500 µL, 5 mmol), CuI (1.9 g, 10
mmol) and Et N (1.4 mL, 10 mmol) were sequentially added and stirred for 30 min at 0 °C.
3
To this mixture, degassified solution of nitrones (2-4, 2.5 mmol) in DMF (20 mL) was added
and stirred for 24 h at room temperature under argon. The mixture was then poured in water
and filtered through celite. The celite bed was thoroughly washed with EtOAc. The combined
filtrate was taken in a separatory funnel and the EtOAc layer was repeatedly washed with
satd. NH Cl and brine and dried over Na SO . The solvent was removed and the residue upon
4
2
4
flash column chomatography (Neutral alumina for 5, Si-gel for 6, 7) using 1:1 PE/EA as
eluent afforded the target β-lactams. Spectral characteristics of the purified compounds are
mentioned below:
Trans [N-phenyl-3-ethoxycarbonyl-4(4-pyridyl)]-2-azetidinone (5)
1
4

ACCEPTED MANUSCRIPT
Yield: 70%; State: red gummy liquid; δ (CDCl , 200 MHz): 8.61-8.58 (2H, d, J = 6 Hz),
H
3
7
.29-7.01 (7H, m), 5.31 (1H, d, J = 2.6 Hz), 4.25 (2H, q, J = 7.1 Hz), 3.94 (1H, d, J = 2.6
Hz), 1.29 (3H, t, J = 7.2 Hz); δ (CDCl , 50 MHz): 165.6, 158.4, 150.7, 145.3, 136.6, 129.2,
C
3
+
1
2
24.7, 120.9, 116.9, 62.9, 62.4, 56.0, 14.1. HRMS (ES+): Calcd. For C H N O +H
17 16 2 3
97.1239, found 297.1344.
Trans [N-phenyl-3-ethoxycarbonyl-4(2-thienyl)]-2-azetidinone (6)
Yield: 75%; State: reddish-yellow gummy liquid; δ (CDCl , 200 MHz): 7.37-6.98 (8H, m),
H
3
5
7
1
3
.62 (1H, d, J = 2.6 Hz), 4.30 (2H, q, J = 7.2 Hz), 4.14 (1H, d, J = 2.6 Hz), 1.33 (3H, t, J =
.2 Hz); δ (CDCl , 50 MHz): 166.0, 159.0, 140.0, 137.0, 129.2, 127.4, 126.8, 126.5, 124.6,
C
3
+
17.3, 64.5, 62.3, 53.6, 14.2. HRMS (ES+): Calcd. forC H NO S+H 302.0851, found
1
6
15
3
02.0867.
Trans [N-phenyl-3-ethoxycarbonyl-4(2-furyl)]-2-azetidinone (7)
Yield: 73%; State: Yellow gummy liquid; δ (CDCl , 400 MHz): 7.41-7.25 (5H, m), 7.10-
H
3
7
2
1
.06 (1H, m), 6.54-6.53 (1H, m), 6.39-6.37 (1H, m), 5.37 (1H, d, J = 2.8 Hz), 4.32 (1H, d, J =
.4 Hz), 4.28 (2H, q, J = 7.2 Hz), 1.33 (3H, t, J = 7.2 Hz); δ (CDCl , 100 MHz): 166.2,
C
3
59.1, 148.7, 143.7, 137.4, 129.2, 124.6, 117.0, 110.8, 110.7, 62.3, 60.5, 50.9, 14.3. HRMS
+
(
ES+): Calcd. forC H NO +H 286.1079, found 286.1123.
1
6
15
4
General procedure of the ester hydrolysis for the synthesis of 8-10
To a solution of ester (5-7, 0.5 mmol) in methanol (20 mL) at 0°C, 0.1 N LiOH (5
mL) was slowly added and the mixture was stirred at 20°C for 6 h. It was then carefully
acidified using 0.5 N HCl at ice-cold condition. The mixture was partitioned between EtOAc
and water (50 mL each). The aqueous layer was back extracted with EtOAc (3 x 50 mL).
Combined organic layers were dried over Na SO and evaporated under reduced pressure to
2
4
produce the target compounds which were purified by repeated precipitation from EtOAc-
hexane. Spectral characteristics of the purified compounds are mentioned below:
Trans [N-phenyl-4(4-pyridyl)]-2-azetidinone-3-carboxylic acid (8)
Yield: 60%; State: red sticky mass; δ (Acetone-d , 400 MHz): 8.63 (2H, m, Ar-H), 7.50
H
6
(2H, d, J = 4.4 Hz, Ar-H), 7.35-7.27 (4H, m, Ar-H), 7.12-7.10 (1H, m, Ar-H), 5.45 (1H, d, J
=
2.8 Hz, H-4), 4.23 (1H, d, J = 2 Hz, H-3); δ (Acetone-d , 100 MHz): 167.4, 159.8, 151.0,
C
6
1
5

ACCEPTED MANUSCRIPT
1
47.0, 138.1, 130.0, 125.2, 122.5, 117.7, 63.7, 56.9; HRMS (ES+): Calcd. For
+
C H N O +H 269.0926, found 269.0948.
15
12
2
3
Trans [N-phenyl-4(2-thienyl)]-2-azetidinone-3-carboxylic acid (9)
Yield: 65%; State: reddish yellow sticky mass; δ (Acetone-d , 400 MHz):7.45-7.27 (6H, m),
H
6
7
1
.08-7.03 (2H, m), 5.74 (1H, bs), 3.67 (1H, bs); δ (Acetone-d , 100 MHz): 170.9, 168.0,
C 6
41.6, 138.3, 130.0, 128.3, 128.2, 127.3, 125.1, 117.9, 60.6, 52.4; HRMS (ES+): Calcd. For
+
C H NO S+Na 296.0357, found 296.0400.
14
11
3
Trans [N-phenyl-4(2-furyl)]-2-azetidinone-3-carboxylic acid (10)
Yield: 68%; State: dark yellow sticky mass; δ (CDCl , 400 MHz): 9.03 (1H, bs), 7.40-7.24
H
3
(5H, m), 7.10-7.06 (1H, m), 6.55 (1H, d, J = 2.8 Hz), 6.37 (1H, q, J = 2 Hz), 5.39 (1H, d, J =
2
1
2
Hz), 4.39 (1H, d, J = 2.4 Hz); δ (CDCl , 100 MHz): 170.1, 159.1, 148.1, 143.8, 137.1,
C 3
+
29.2, 124.9, 117.2, 110.9, 110.9, 60.0, 51.1; HRMS (ES+): Calcd. For C H NO +Na
1
4
11
4
58.0766, found 258.0804.
Synthesis of Trans [3-(hydroxymethyl)-1-phenyl-4-(4-pyridyl)]-2-azetidinone (11)
To a solution of 8 (70 mg, 0.24 mmol) in methanol (10 mL), NaBH (19 mg, 0.5
4
mmol) was added pinch-wise and stirred for 30 min at room temperature. After completion of
the reaction as indicated by TLC, the reaction mixture was quenched with satd. NH Cl
4
solution and extracted with EtOAc. The organic layer was washed by brine, dried over
Na SO and evaporated under reduced pressure. The residue upon flash column
2
4
chromatography (neutral alumina, 2:1 PE-EA as eluent) afforded the pure compounds.
Spectral characteristics of the purified compounds are mentioned below:
Yield: 72%; State: red sticky mass; δ (CDCl , 400 MHz): 8.62 (2H, bs), 7.32-7.24 (6H, m),
H
3
7
.11-7.07 (1H, m), 5.06 (1H, d, J = 2.4 Hz), 4.18 (1H, dd, J = 12 Hz, 4.8 Hz), 4.05 (1H, dd, J
12 Hz, 4 Hz), 3.37 (1H, t, J = 5.2 Hz), 3.28 (1H, bs); δ (CDCl , 100 MHz): 164.9, 150.5,
=
C
3
1
47.3, 137.1, 129.4, 124.6, 121.1, 117.0, 62.2, 58.7, 56.2; HRMS (ES+): Calcd. For
+
C H N O +H 255.1134, found 255.1106.
15
14
2
2
General procedure of EDC coupling reaction for the synthesis of 12-14
1
6

ACCEPTED MANUSCRIPT
In dry DCM (10 mL), compound 8-10 (0.2 mmol), EDC-Cl (26 mg, 0.2 mmol), HOBt
(27 mg, 0.2 mmol) were added and stirred at 0 °C for 15 min. Isoniazid (23.3 mg, 0.17 mmol)
and DMAP (24 mg, 0.2 mmol) were then added and the mixture was stirred at room
temperature for 16 h. The mixture was then partitioned between water and DCM (50 mL
each) and washed with brine. The organic layer was dried with Na SO and evaporated under
2
4
reduced pressure to leave a residue from which the target compounds were purified by flash
column chromatography (neutral alumina) using 10:1 DCM-methanol as eluent. Spectral
characteristics of the purified compounds are mentioned below:
Trans [N'-isonicotinoyl-2-oxo-1-phenyl-4-(4-pyridyl)]-azetidine-3-carbohydrazide (12)
Yield: 75%; State: reddish sticky mass; δ (Acetone-d , 400 MHz): 8.77 (2H, d, J = 5.6 Hz),
H
6
8
.64 (2H, d, J = 4.6 Hz), 7.82 (2H, d, J = 6 Hz), 7.50 (2H, d, J = 5.6 Hz), 7.33-7.31 (4H, m),
.13-7.09 (1H, m), 5.47 (1H, d, J = 2.4 Hz), 4.26 (1H, d, J = 2.4 Hz); δ (Acetone-d , 100
7
C
6
MHz): 164.8, 164.8, 160.7, 151.4, 151.3, 146.8, 140.4, 138.1, 130.1, 125.2, 122.2, 122.0,
+
1
17.7, 63.0, 56.8; HRMS (ES+): Calcd. For C H N O +H 388.1410, found 388.1417.
2
1
17
5
3
Trans [N'-isonicotinoyl-2-oxo-1-phenyl-4-(2-thienyl)]-azetidine-3-carbohydrazide (13)
Yield: 80%; State: reddish-yellow sticky mass; δ (CDCl , 400 MHz): 8.62 (2H, d, J = 4.4
H
3
Hz), 7.62 (2H, d, J = 5.2 Hz), 7.27-6.92 (8H, m), 5.75 (1H, d, J = 1.6 Hz), 4.36 (1H, d, J =
2
1
3
.4 Hz); δ (CDCl , 100 MHz): 163.6, 163.2, 160.8, 150.4, 139.4, 138.6, 136.6, 129.2, 127.5,
C 3
+
27.4, 126.7, 125.1, 121.4, 117.5, 63.0, 53.4; HRMS (ES+): Calcd. For C H N O S+H
2
0
16
4
3
93.1021, found 393.1018.
Trans [N'-isonicotinoyl-2-oxo-1-phenyl-4-(2-furyl)]-azetidine-3-carbohydrazide (14)
Yield: 79%; State: reddish-yellow sticky mass; δ (Acetonitrile-d , 400 MHz): 8.57 (2H, d, J
H
3
=
4.4 Hz), 7.52 (2H, d, J = 6 Hz), 7.37 (1H, s), 7.22-7.14 (4H, m), 6.98-6.94 (1H, m), 6.56
(1H, d, J = 3.2 Hz), 6.31-6.29 (1H, m), 5.30 (1H, d, J = 3.2 Hz), 4.29 (1H, d, J = 2.4 Hz); δC
(
Acetonitrile-d , 100 MHz): 165.2, 165.2, 161.1, 151.4, 149.5, 144.9, 140.1, 138.0, 130.1,
3
+
1
3
25.5, 122.1, 117.7, 112.1, 111.8, 59.9, 51.4; HRMS (ES+): Calcd. For C H N O +H
20 16 4 4
77.1250, found 377.1251.
Synthesis
of
Trans
[N'-benzoyl-2-oxo-1-phenyl-4-(4-pyridyl)]-azetidine-3-
carbohydrazide (16)
1
7

ACCEPTED MANUSCRIPT
To a solution of compound 8 (0.2 mmol) in dry DCM, HOBt (27 mg, 0.2 mmol) and
EDC-Cl (26 mg, 0.2 mmol) were added and stirred for 15 min at 0 °C. Benzhydrazide (7, 0.2
mmol) and Et N (20 µL, 0.2 mmol) were then added and stirred for 12 h at room temperature.
3
It was then partitioned between DCM and water, the organic layer was washed with brine.
The organic layer was dried over Na SO and evaporated at reduced pressure. The residue
2
4
upon flash column chromatography (Si-gel, 10:1 DCM-methanol as eluent) furnished the
target compounds. Spectral characteristics of the purified compounds are mentioned below:
Yield: 70%; State: red-yellow sticky mass; δ (Acetone-d , 400 MHz): 8.64 (2H, d, J = 4.4
H
6
Hz), 7.98 (2H, d, J = 7.6 Hz), 7.61-7.49 (5H, m), 7.32-7.31 (4H, m), 7.10 (1H, m), 5.51 (1H,
d, J = 2.4 Hz), 4.17 (1H, d, J = 2.8 Hz); δ (Acetone-d , 100 MHz): 167.3, 167.0, 159.9,
c
6
1
51.0, 147.1, 138.1, 133.9, 132.7, 130.1, 129.3, 128.3, 125.2, 122.5, 117.7, 63.6, 56.9;
+
HRMS (ES+): Calcd. For C H N O +H 387.1457, found 387.1507.
22
18
4
3
Protein purification and Inhibition kinetics
FabG4 purification and inhibition kinetics: Detailed procedure of protein purification had
2
0
been described elsewhere. The inhibition studies were carried out by measuring the
decrease of OD340nm due to the conversion of NADH to NAD+ in Evolution™ 300 UV–
Visible spectrophotometer (Thermo Fisher scientific) at 25 °C. The assay was done in
HEPES buffer (50 mM, pH=7.4) in a volume of 500 µL. The acetoacetyl-CoA and β-NADH
were purchased from sigma and dissolved in miliQ water to desired stocks concentrations.
The inhibitor compounds were dissolved in HPLC grade methanol to a primary stock of 10
mM concentration. From that various diluted stocks were prepared using methanol and
exactly 25 µL of inhibitor solution was added to 500 µL reaction mixture to maintain the
same percentage of methanol (5%) in every assay. This 5% methanol was also added to the
controls to nullify the effect of methanol on inhibition kinetics. The typical reaction mixture
contained 25 µL of inhibitor solution in methanol, 0.5 mM acetoacetyl-CoA, 0.2 mM β-
NADH, 1 ꢁM of FabG4 and remaining HEPES buffer in the final volume of 500 ꢁL. A
mixture of 25 µL inhibitor in methanol, 0.5 mM acetoacetyl-CoA and 1 ꢁM FabG4 in 500 µL
buffer was used for baseline correction (negative control) before each assay; while mixture of
2
5 µL methanol (without inhibitor), 0.5 mM acetoacetyl-CoA, 1ꢁM FabG4 and 0.2mM β-
NADH in 500 µL buffer was used as positive control. The reaction was initiated by the
addition of acetoacetyl-CoA. The decrease in absorbance was recorded in every 2 min
interval. IC values were determined by varying inhibitor concentrations until full inhibition
5
0
1
8

ACCEPTED MANUSCRIPT
occurred. Enzyme activities were measured with varying NADH concentrations at three
different concentrations of inhibitor to determine the mode of inhibition. The IC values
5
0
were calculated graphically from dose-response plots. Modes of inhibition were determined
from Lineweaver-Burk plots.
HtdX purification and inhibition kinetics: The details of HtdX purification has been
1
8
described previously from this group.
Inhibition kinetics were performed
spectrophotometrically at 263 nm by using crotonoyl-CoA as substrate in Evolution™ 300
UV–Visible spectrophotometer at 25 °C. The assays were done in HEPES buffer (50 mM,
pH=7.5) in presence of 100 mM NaCl with a final volume of 500 µL. The crotonoyl-CoA
was purchased from sigma and dissolved in miliQ water to desired stock concentration. The
inhibitor solutions were prepared as described in FabG4 inhibition kinetics section and
similarly 25 µL of inhibitor solution was added to 500 µL reaction mixture to maintain the
same percentage of methanol (5%) in every assay. This 5% methanol was also added to the
controls to nullify the effect of methanol on inhibition kinetics. The typical reaction mixture
contained 25 µL of inhibitor solution in methanol, 10 µM crotonoyl-CoA, 100 nM HtdX and
remaining buffer in the final volume of 500ꢁL. A mixture of 25 µL inhibitor in methanol and
1
00 nM HtdX in 500 µL buffer was used for baseline correction (negative control) before
each assay; while mixture of 25 µL methanol (without inhibitor), 10 µM crotonoyl-CoA and
00 nM HtdX in 500 µL buffer was used as positive control. The reaction was initiated by the
1
addition of crotonoyl-CoA. The decrease in absorbance was recorded in every 2 min interval.
IC values were determined by varying inhibitor concentrations until full inhibition occurred.
5
0
Mode of inhibition was measured by varying crotonoyl-CoA concentrations at three different
concentrations of inhibitor. The IC values were calculated graphically from dose-response
5
0
plots. Modes of inhibition were determined from Lineweaver-Burk plots.
Methods of ITC study
FabG4-12 and HtdX-13 ITC studies were carried out in Microcal ITC200 instrument at
2
0
5 ˚C. Reference power of 5 µcal / sec, stirring speed of 500 rpm, duration of each injection
.8 sec, filter period 5 sec with 120 sec spacing were selected as parameters for these studies.
FabG4 stock solution was prepared in 50mM HEPES buffer (pH 7.4); while HtdX solution
was prepared in HEPES buffer (50 mM, pH=7.5) in presence of 100 mM NaCl. Compound
stock solutions were prepared in 5 % methanol-respective buffers. This extra methanol was
also added to the sample cell to avoid large heat change due to solvent mismatch (Getting
1
9

ACCEPTED MANUSCRIPT
Started, MicroCal ITC200). All stock solutions were degasified properly before performing
experiments. Finally, 4.5 µM FabG4 solution was taken in sample cell and titrated with 500
µM compound 12 solution. Similarly, 10 µM HtdX solution (in sample cell) was titrated
against 500 µM compound 13 solution. Separate buffer-ligand titrations were carried out in
each case as reference runs by taking buffer in the sample cell. These reference values were
subtracted from the protein-ligand titrations to nullify heat of dissolution. One injection of 0.4
µL followed by nineteen injections of 2 µL of ligand solutions were titrated into protein
solutions in both cases. The data were solved using MicroCal, LLC ITC 200 software.
Methods of CD study
CD measurements were carried out on a Jasco-815 automatic recording
o
spectrophotometer, using a path length of 10 mm at 25 C. The spectra were recorded in the
range of 195–240 nm with a scan rate of 50 nm / min and a response time of 1 s. For baseline
correction, CD spectra of buffer (10 mM tris buffer of pH 6.8) containing 50 µM ligands
were collected and were subtracted from each sample spectra. The CD spectra of protein-
ligand complexes were collected at protein to ligand molar ratio 1:5. The ligand
concentrations were maintained at 50 µM with the protein concentration at 10 µM. Secondary
3
7
structures analysis were carried out using DICHROWEB, an online server for protein
secondary structure analysis from CD spectroscopic data.
Methods of MIC assay
2
Mycobacterium smegmatis mc 155 strain was grown in Middlebrook 7H9 broth with
0
.2 % glycerol and 0.05 % Tween 80 for 20 hours at 37 °C with shaking at 120 rpm till the
cells reached mid-logarithmic phase (OD595 ~ 0.5). Reference compound isoniazid (INH) was
dissolved in distilled water to primary stock solution (5 mg/mL) and serially diluted in MB
7
H9 media to working stock solution (0.5 mg/mL). All synthesized inhibitors were dissolved
in DMSO to an initial stock of 5 mg/mL and two fold diluted in MB 7H9 media to working
stock solution (0.5 mg/mL). Resazurin sodium salt was purchased from Sisco Research
Laboratories, India. 0.5 % (w/v) stock solution of resazurin was prepared in distilled water,
filter sterilized and diluted to 0.02 % in distilled water.
The inoculum was omitted from row A (negative control), only 50 µL of media were
added to each well. Inhibitor and isoniazid were omitted from row B (growth control), 50 µL
media and 50 µL of diluted culture (OD595 = 0.0005) were added to each well. The media,
2
0

ACCEPTED MANUSCRIPT
reference compound isoniazid (INH) and culture were added in row C (positive control). In
rows D-F, compound 12, 13 and 14 were added instead of INH along with culture and media.
The total volume in each well was 100 µL. After 24 hours of post-drug incubation at 37 °C,
3
0 µL of 0.02 % resazurin solution was added to each well and incubated again for 40 mins at
7 °C. Color change from blue (resazurin) to pink (resorufin) indicated the growth of
3
bacteria. The lowest concentrations of drugs which prevented such colour change were
recorded as MIC. The assay was repeated twice and average of both the experiments was
calculated to find the MIC values.
Methods of biofilm assay
2
Biofilm of M. smegmatis mc 155 was grown in 6-well flat bottomed plate by
inoculating 30 uL (1%) of planktonic culture (growth of M. smegmatis planktonic culture
already described in previous method) in 3 mL Sauton’s minimal media (without Tween-80).
Well 1 was used as negative control without any inhibitor, while we added inhibitor 12 at
-1
increasing concentration of 5, 10, 20, 50 and 100 µg mL respectively in well 2 to well 6.
The plate was sealed with grease, wrapped up by parafilm and incubated at 37 °C without
shaking for 4 weeks. Biofilm formation was easily detected using naked eye. The lowest
concentration of inhibitor that prevented biofilm formation was recorded.
SUPPLEMENTARY DATA
1
13
H and C NMR spectra of synthesized compounds, mass spectra and HPLC traces of
found dual inhibitors, inhibition kinetics data, ITC data, CD data, additional docking studies
and MIC plate images are included in supporting information.
ACKNOWLEDGMENT
DRB thanks the Council of Scientific and Industrial Research (CSIR) for the
fellowship. Central Research Facility and Department of Chemistry, IIT Kharagpur are
thanked for providing all of the instrumental facilities. AB and AKD gratefully acknowledge
the support of DBT and for providing the necessary research grant. AB and AKD also
acknowledge IIT Kharagpur for SGBSI grant. AB is also grateful to DST for the JC Bose
National Fellowship.
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FIGURE CAPTIONS
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Figure 1: A. The genomic arrangement of Rv0242c (FabG4) and Rv0241c (HtdX) in
conserved putative fatty acid metabolizing operon (OperonDB, confidence = 84, n = 57); B.
Basic functions of FabG4 and HtdX; FabG4 and HtdX are involved in two consecutive steps
of fatty acid metabolism.
Figure 2: NADH bound FabG4 structure with close view of catalytic tetrad (Asn319, Ser347,
Tyr360, Lys364) and catalytic loops (I and II). NADH binds at wide major portal (green),
minor portal (magenta) is narrow.
Figure 3. (at top) Alignment of amino acid sequence of the catalytic loop of M. tuberculosis
HtdX (3WEW) with C. tropicalis 2-Enoyl-CoA hydratase 2 (1PN2), peroxisomal
dehydrogenase/hydratase 2(1S9C), P. capsici Mao-C like dehydratase (3KH8) and M.
tuberculosis HadB. Red boxes indicate highly conserved residues;
(at below) Overall structure of HtdX with close view of catalytic loop. Active site residues
Asp162 and His167 are inside the active site tunnel, the outer hydrophobic lid consists of
Ile171, Leu175, Phe176 and Phe178. Vacuum electrostatics contact potential of catalytic loop
(from the same face) shows that the deep of catalytic tunnel is highly negative charged (red).
Figure 4: Eight designed compounds as possible inhibitors of FabG4 and HtdX.
Figure 5: ORTEP image of 14 (CCDC number 1042896)
Figure 6: A. FabG4-12 docking results: compound 12 binds at major portal of FabG4 and
interacts with Tyr360 (catalytic residue), Lys364 (catalytic residue), Gly297, Ala296,
Asp244; B. HtdX-13 docking results: compound 13 binds near catalytic loop and interacts
with Gly185, Phe178, His167 (catalytic residue) and Asp162 (catalytic residue). More images
are included in supporting information.
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TABLES.
Table 1: Inhibition profiles of designed and synthesized compounds
IC against
IC against
50
IC against
50
IC against
50
5
0
Compound
Mtb-FabG4
in µM)
Mtb-HtdX
(in µM)
Compound
Mtb-FabG4
(in µM)
Mtb-HtdX
(in µM)
(
N
N
H
No
Inhibition
H
6
4.3±1
H
N
15.2±0.5
26.1±1.5
22.3±0.5
10.3±0.8
17.6±0.9
O
C
H
N
H
N
N
H
C
O
8
O
HOOC
N
N
12
O
S
S
H
H
No
No
Inhibition
O
C
H
N
H
N
H
N
Inhibition
N
H
C
O
9
O
HOOC
O
13
O
O
H
H
No
Inhibition
No
Inhibition
O
H
H
N
2
7.6±1
C
N
H
N
N
C
O
H
O
N
HOOC
10
1
4
O
N
N
H
H
No
Inhibition
No
Inhibition
6
66.1±20
O
C
H
H
N
39.9±2
H
N
N
N
H
C
O
11
O
1
6
O
OH
Table 2: Mode of bindings, binding constants and thermodynamic parameters from FabG4-
2 and HtdX-13 titrations
1
Binding constant
K
Thermodynamic parameters
Titration
FabG4-12
HtdX-13
Mode of Binding
ꢀH
ꢀS
ꢀG
-1
(Mol )
(
Kcal/Mol)
(Cal/Mol/K)
(Kcal/Mol)
Sequential binding
With number of
binding
5
3
K = 2.98 x 10
ꢀH = -15.7
ꢀS = -27.8
ꢀG = - 7.4
1
1
1
1
2
3
K = 1.15 x 10
2
ꢀH = -4.8x10
ꢀS =-1.6x10
ꢀG = -4.0
2
2
2
Sites (N) = 2
5
One site binding
K= 4.21 x 10
ꢀH = -66.2
ꢀS = -196
ꢀG = - 7.8
2
8