In silico designing of domain B selective gyrase inhibitors for effective treatment of resistant tuberculosis

Article abstract of DOI:10.1016/j.tube.2018.08.005

One of the major mechanisms followed by the therapeutic agents to target the causative organism of TB, Mycobacterium tuberculosis, involves disruption of its DNA replication cycle. The process of replication involves two steps, i.e., breakage and reunion of DNA at gyrase A (GyrA) domain and ATP hydrolysis at gyrase B (GyrB) domain, both occur simultaneously. Current therapy for multi-drug resistant TB (MDR-TB) involves FDA approved, fluoroquinolone-based antibiotics, which act by targeting replication process at GyrA domain. However, resistance against fluoroquinolones due to mutations in the GyrA domain has limited the use of this therapy and shifted the focus of research community on GyrB domain. Thus, in the present study novel chemotherapeutic agents for resistant TB were designed by exploring GyrB domain using in silico techniques. Pharmacophore model of GyrB domain was employed for screening an In-house database. Followed by cross-screening via a qualitative Hip-Hop pharmacophore model for GyrA to remove non-selective gyrase B inhibitors. Further molecular dynamics simulations and MM-GBSA calculations were performed to determine stability and binding affinity of the screened molecules. These analyses resulted in five putative oxindole based selective GyrB domain inhibitors, which were synthesized and evaluated for anti-tubercular activity against M. tuberculosis H₃₇Rv strain. Two compounds exhibited significant anti-TB activity, whereas other three compounds were found to be outliers of the in silico study.

Full text of DOI:10.1016/j.tube.2018.08.005

Accepted Manuscript
In silico designing of domain B selective gyrase inhibitors for effective treatment of
resistant tuberculosis
Aanchal Kashyap, Pankaj Kumar Singh, Om Silakari
PII:
S1472-9792(18)30189-6
10.1016/j.tube.2018.08.005
YTUBE 1739
DOI:
Reference:
To appear in: Tuberculosis
Received Date: 4 May 2018
Revised Date: 22 July 2018
Accepted Date: 6 August 2018
Please cite this article as: Kashyap A, Singh PK, Silakari O, In silico designing of domain B selective
gyrase inhibitors for effective treatment of resistant tuberculosis, Tuberculosis (2018), doi: 10.1016/
j.tube.2018.08.005.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
In Silico designing of Domain B Selective Gyrase Inhibitors for Effective Treatment of
Resistant Tuberculosis
Aanchal Kashyap, Pankaj Kumar Singh, Om Silakari
Molecular Modeling Lab (MML), Department of Pharmaceutical Sciences and Drug Research,
Punjabi University, Patiala, Punjab, 147002, India.
*Corresponding authors E-mail: omsilakari@gmail.com; Tel: +91-9501542696; Fax: +91-
1752283075

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Abstract
One of the major mechanisms followed by the therapeutic agents to target the causative organism
of TB, Mycobacterium tuberculosis, involves disruption of its DNA replication cycle. The
process of replication involves two steps, i.e., breakage and reunion of DNA at gyrase A (GyrA)
domain and ATP hydrolysis at gyrase B (GyrB) domain, both occur simultaneously. Current
therapy for multi-drug resistant TB (MDR-TB) involves FDA approved, fluoroquinolone-based
antibiotics, which act by targeting replication process at GyrA domain. However, resistance
against fluoroquinolones due to mutations in the GyrA domain has limited the use of this therapy
and shifted the focus of research community on GyrB domain. Thus, in the present study novel
chemotherapeutic agents for resistant TB were designed by exploring GyrB domain using in
silico techniques. Pharmacophore model of GyrB domain was employed for screening an In-
house database. Followed by cross-screening via a qualitative Hip-Hop pharmacophore model
for GyrA to remove non-selective gyrase B inhibitors. Further molecular dynamics simulations
and MM-GBSA calculations were performed to determine stability and binding affinity of the
screened molecules. These analyses resulted in five putative oxindole based selective GyrB
domain inhibitors, which were synthesized and evaluated for anti-tubercular activity against M.
tuberculosis H37Rv strain. Two compounds exhibited significant anti-TB activity, whereas other
three compounds were found to be outliers of the in silico study.

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1 Introduction
Tuberculosis (TB) is still a serious major health concern that has existed from millennia.
Tuberculosis is characterized by necrotizing granulomatous inflammation usually in the lungs.
An aetiologic agent, M. tuberculosis is responsible for this infectious communicable disease. It is
a slow growing, acid fast bacillus that is capable of persisting in a semi dormant state of
replication and likely to reactivate when the host immune response is compromised (Zhang
2004). It was ranked above HIV/AIDS as one of the top 10 leading cause of death from an
infectious disease in 2015. Since this disease is contagious in nature, immunocompromised
patients are at a higher risk of developing it. About 1.5 million TB deaths were estimated in 2015
of which around 0.4 million deaths were estimated in immunocompromised patients. About 10.4
million new cases of TB are also estimated. According to WHO report 2016, India is listed in the
top 20 high TB burden countries and top 30 HIV/TB and Resistant TB countries (Organization
2015). However, occurrence of drug resistance, particularly multidrug-resistant (MDR), which
involves resistance to rifampicin and isoniazid, has led to the failure of first line therapy
(Pourakbari, Mamishi et al. 2016). For such cases of resistance and failure of first line therapy,
FDA- approved fluoroquinolones, aminoglycosides (kanamycin, amikacin) and capreomycin are
used as a second line therapy. Fluoroquinolones, an important second line drug, act by binding
in the GyrA domain of the DNA gyrase enzyme in M. tuberculosis. DNA gyrase is a sole
topoisomerase II in M. tuberculosis which regulates relaxation of negative supercoils or
introduction of the either negative or positive supercoils into DNA along with catenation and
decatenation of circular DNAs. Gyrase is a validated target of anti-TB chemotherapy especially
in case of MDR-TB (Collin, Karkare et al. 2011). In 2012, Chopra and his research group
reported aminobenzimidazole 1 as GyrB inhibitor which exhibited significant bactericidal
activity establishing cidal activity nature of GyrB inhibition (Chopra, Matsuyama et al. 2011).
M. tuberculosis DNA gyrase is a heterotetramer (A2B2) consisting of two domains, gyrase A
(encoded by gyrA gene) and gyrase B (encoded by gyrB gene) which are involved in maintaining
the topology of DNA. Domain A is involved in the breakage and reunion of the DNA, whereas
domain B promote ATP hydrolysis (Mdluli and Ma 2007). Although fluoroquinolones are
available as approved GyrA inhibitors for MDR-TB, occurrence of acquired mutations in QRDR
(Quinolone Resistance Determining Region) of GyrA results in development of resistance.

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QRDR is a small conserved cluster of about 40 amino acids near the catalytic tyrosine, involved
in scission of DNA (Takiff, Salazar et al. 1994). To counter such resistance mechanism different
approaches are being explored by researchers. Out of all, targeting the energy-supplying domain
B of gyrase appears to be a lucrative and effective option. Till date, there is only one approved
antibiotic, novobiocin, an aminocoumarin natural product, which target gyrase B subunit.
Originally novobiocin was approved to treat methicillin-resistant Staphylococcus aureus
(MRSA) infections but was withdrawn from the market because it suffered from poor
pharmacological properties such as low efficacy and raised safety concerns. Although GyrB
domain has been previously demonstrated as bactericidal drug target, no significant drug
designing studies have been conducted against this target (Kaur, Agarwal et al. 2009).
Because of the resistance to fluoroquinolone based second line therapy, we laid efforts to design
selective inhibitors of GyrB domain. Focus has been given to ensure the selectivity of the
designed molecules, so that resistant TB could be targeted in a dose effective manner. Moreover,
as GyrB is an established anti-TB target, the designed inhibitors are expected to be effective in
both non-resistant TB as well as resistant TB. Hence, in our search for inhibitors of GyrB as anti-
TB agents, pharmacophore model of GyrB, to screen the in house database along with hip-hop
model of GyrA, was utilized. The selective GyrB inhibitors were also subjected to docking
analysis, MM-GBSA and MD simulations, to validate the selectivity and potency the molecules
for GyrB domain. Followed by synthesis and evaluation of their anti-TB potential.
2. Material and methods
2.1. In-silico study
A ligand based pharmacophore model for a specific set of molecules provides essential features
that are necessary to produce their respective biological activity. Such model can then be used to
screen and identify novel molecules with structural features suitable for inhibitory potential
against desired target (Singh and Silakari 2017). Thus, previously developed 3D pharmacophore
model for GyrB domain was utilized as the screening tool against an in-house database of
oxindole derivatives. The oxindole derivatives retrieved from screening were further subjected to
molecular docking analysis and molecular dynamics (MD) simulations to predict their binding
orientations and conformations. Molecular docking is a technique to elucidate the probable

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interactions between the designed compounds in the active site of the target protein. However, as
docking tool utilizes proteins as rigid structure and is considered ‘rough’ method, it was followed
by MD (molecular dynamics) simulations Docking studies allows to characterize the ligand
conformation along with its position and orientation into the active site of the target protein. The
CDOCKER tool of DS (Discovery Studio) was used to perform and analyze docking interactions
of the designed compounds with the ATP binding site in the GyrB domain. The poses generated
for each ligand were selected on the basis of the CDOCKER interaction energy For the docking
analysis of GyrB domain, among two available structures i.e. 4B6C and 4BAE, 4B6C co-
crystallized with aminopyrazinamide was selected on the basis of better resolution. Similarly for
the docking analysis of GyrA domain, among available eight crystal structures, 5BTA co-
crystallized with moxifloxacin was selected on the basis of cross docking analysis (Singh and
Silakari 2018).
The stability of the designed molecules and protein complex was then analyzed by performing
MD simulations for the period of 10 ns using academic version of “Desmond” program. MD
simulations are theoretical calculations considering the flexible nature of protein, similar to that
present in the physiological system (van Gunsteren and Berendsen 1990). The solvent system
was prepared using TIP3P with orthorhombic water box shape. The system was neutralized by
adding Na+ ions and salt concentration was set at 0.15M corresponding to physiological system.
The system hence prepared was subjected to molecular dynamics simulations using NPT
ensemble where temperature and pressure were maintained as 310K and 1bar using Nose-Hoover
Chain as thermostat and Martyna-Tobais-Klein as barostat, respectively. The simulations were
run for a period of 10 ns with time step of 1.0 fs (Shivakumar, Williams et al. 2010). Finally the
binding affinity for both the domains was predicted by calculating MM-GBSA (Molecular
Mechanics with generalized Born and Surface area solvation) score available with Schrodinger
software. The MM-GBSA calculations are generally performed to estimate the free binding
energy of the ligand with that of target macromolecules (Genheden and Ryde 2015). Further to
evaluate the effect of acquired mutations in GyrB on the binding affinity of the designed
molecules, the GyrB mutant bearing most common N499D mutation was build and
corresponding binding affinity for the designed molecules was calculated by performing MM-
GBSA calculations (Piton, Petrella et al. 2010).

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2.2.
Chemistry
Commercially available reagents and solvents were used for the synthetic purpose. Pre-coated
TLC plates were used to monitor the reactions. Column chromatography was carried out on
silica gel 60-120 mesh for the purification and separation of the compounds using a combination
of ethyl acetate and petroleum ether. Melting points were obtained in open capillary tubes on a
melting point apparatus. The IR spectra of the molecules were recorded on a FT-IR
1
spectrophotometer, using KBr pellet. H NMR and 13C NMR spectra of the molecules were
generated on a Bruker spectrophotometer at 400MHz and 100MHz (TMS as internal standard),
respectively using CDCl3 or DMSO as solvent. The designed compounds were synthesized
according to the Scheme 1.
2.2.1. Procedure for the synthesis of 3-(4-Hydroxybenzylidene)indolin-2-one (A)
2-Oxindole (0.9 mmol) was treated with p-hydroxy benzaldehyde (1mmol) in the presence of 1
ml of piperidine as a catalyst and refluxed for 4-5 hrs (Scheme 1). The completion of reaction
was monitored using TLC. Upon completion, the solvent was evaporated to obtain crude
product. The crude product was purified via recrystalization in ethanol.
2.2.2. Synthesis of variedly substituted 2-aryl ester substituted oxindole derivatives (A1-
A5).
Compound A (1mmol) was suspended in DCM, placed in a round bottom flask. About 2 ml of
triethylamine was added as a base. Different benzoyl chlorides (1mmol) were added to this basic
solution and stirred for 3-4 hr at room temperature. The reaction progress was monitored by
TLC. After completion of the reaction, then solvent was evaporated to obtain crude product. It
was subjected to column chromatography with chloroform:methanol as solvent system to obtain
desired products (A1-A5). The scheme for synthesized compounds is shown in Scheme 1.
<Scheme 1>
2.3. In-vitro antitubercular activity of the synthesized compounds (Lowenstein-Jensen
method)

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Antitubercular activity was determined against Mycobacterium tuberculosis H37Rv, employing
Lowenstein-Jensen method with slight modification. 2000 ꢀg/ml dilution of each test compound
was added to liquid Lowenstein-Jensen Medium, followed by sterilization using inspissation
method. A culture of Mycobacterium tuberculosis H37Rv growing on Lowenstein Jensen
medium was harvested in 0.85% saline, in bijou bottles. For all test compounds, a stock solution
of 2000 ꢀg/ml concentration was prepared in DMSO. These tubes were then incubated at 37 °C
for 24 h followed by streaking of M. tuberculosis H37Rv (5 × 104 bacilli per tube). These tubes
were then incubated at 37 °C. Growth of bacilli was seen after 12 days, 22 days and finally 28
days of incubation. Tubes having the compounds were compared with control tubes where
medium alone was incubated with M. tuberculosis H37Rv. The concentration at which no
development of colonies occurred or < 20 colonies was taken as MIC concentration of test
compound (Solankee and Tailor 2016).
3. Result and Discussion
3.1.In-silico analysis
Previously generated ligand based pharmacophore model of GyrB domain and common feature
pharmacophore model for GyrA, built using Discovery studio, were employed to screen and
cross screen an in-house small molecule database (Accelrys 2015). The generated
pharmacophore model for GyrB domain consisted of two hydrogen bond acceptor (HBA), one
hydrogen bond donor (HBD) and one hydrophobic (HY) feature. The mapping of designed
screened compounds on the pharmacophore generated for GyrB domain suggested that the NH
group of the oxindole overlapped on the HBD feature, HY group was occupied by the benzene
ring of the oxindole and the two HBA groups overlapped on the carboxyl group and substitutions
on different benzoyl chlorides (Fig. 1). Molecules possessing significant fit score in mapping via
both pharmacophores were selected for further analysis.
<Figure 1>
This screening yielded a total of five oxindole based compounds, which were then subjected to
docking analysis using co-crystallized 3-D structure of both the target domains followed by MD
simulations and calculation of MM-GBSA score (binding energies). The poses generated for

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each ligand were selected on the basis of the CDOCKER interaction energy and were found to
possess important conserved interactions with the active site residues i.e. Arg82 and Glu56 (Fig.
2).
<Figure 2>
The MD simulations were performed for the period of 10 ns with the time step of 1 fs and
RMSD values of protein and ligand were considered for the analysis of stability of each protein-
ligand complexes. RMSD plot showed complex of the designed molecules with GyrB to be fairly
stable. The interaction diagrams after simulations run time showed maintenance of key
interactions with Arg82 and Glu56. The RMSD curve plotted for the same of the best designed
compound is shown in Fig. 3.
<Figure 3>
The binding affinity of GyrB domain with designed compounds was calculated in terms of MM-
GBSA score using Prime in Schrödinger (Wu, Robertson et al. 2003). The interaction energy
and binding affinity score for both the domains and GyrB mutant is represented in Table 1. The
binding affinity was also calculated for GyrA domain and poor MM-GBSA score for GyrA
domain indicates that the designed compounds are GyrB domain selective. Additionally the
binding affinity for the mutant GyrB was also calculated. The score was comparable to that of
wild type GyrB protein validating that the QRDR region mutations donot alter affinity of the
ATP binding cleft in GyrB domain.
<Table 1>
Chemistry
For synthesizing designed molecules, in the first step, 2-oxindole was treated with p-
hydroxybenzaldehyde via Knoevenagel condensation (Jones 1967), to form 3-(4-
hydroxybenzylidene)indolin-2-one (A) . In the second step, intermediate A was treated with
various benzoyl chlorides in the presence of triethylamine (TEA) using dichloromethane (DCM)
as solvent to yield desired compounds i.e. 3-aryl ester substituted oxindole derivatives (A1-A5)
(Scheme 1). Finally column chromatography was performed to obtain pure derivatives.

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1
Although synthesized compounds are previously reported, they were characterized by IR, H-
NMR and 13C-NMR.
In IR spectrum of synthesized compounds, a broad band appeared in range of 3400-3300 cm-1
was due to N-H stretching vibration. The stretching for ester C=O appeared in range of 1745-
1730 cm-1 showing the coupling of various benzoyl groups over p-hydroxy of benzaldehyde.
Similarly stretching for amide C=O appeared in range of 1590-1690 cm-1. In addition, for
methoxy substituted compounds C-O stretching appeared in range of 1170-1130 cm-1. The IR
spectra of synthesized compounds showed absorption bands characteristics of anticipated
structures.
The synthesis of final compounds was confirmed in 1H-NMR. Almost each spectrum showed
characteristic singlet at chemical shift value of 7.62 for the bridging proton of benzaldehyde.
Similarly, singlet was obtained for one proton present on the benzoyl substitutent at ~7.66 ppm
while rest of the aromatic protons followed conventional pattern and were observed between
~7.82-7.18 ppm. 13C NMR spectral data of compounds showed characteristic peak of amide
carbon in oxindole nucleus at ~168-170 ppm and ester carbon formed after benzoylation at ~164-
166 ppm.
The spectral information of all the designed compounds is given below amd the spectras
obtained are provided in the supplementary.
3-((1-Oxoindolin-2-ylidene)methyl)phenyl benzoate (A1) light yellow solid, Yield: 60%, mp:
186-188oC, Rf 0.51, IR (ν cm-1) 3361 (N-H) (w); 3037 (=C-H str.) (m); 1683 (C=O) (amide) (s),
1730 (C=O) (ester); 1H NMR (400 MHz, CDCl3) δ (ppm): 8.24-8.22 (2H d, J=8 Hz, ArH), 7.93-
7.98 (1H, d, J=8 Hz, ArH), 7.75-7.73 (2H, d, J=8 Hz, ArH), 7.65-7.62 (3H, m, ArH), 7.56-7.47
13
(3H, m, ArH), 7.38-7.36 (2H, d, J= 8 Hz, ArH), 7.11-7.09 (1H, m, ArH); C NMR (100 MHz,
CDCl3) 163.9 (C-16), 163.4 (C-1), 139.2 (C-13), 138.4 (C-8), 138.3 (C-9), 137 (C-10), 131.7 (C-
17), 130.6 (C-18, 22), 129.8 (C-2), 129.6 (C-15, 11), 129.5 (C-20), 127.1 (C-6), 127.0 (C-19,
21), 125.5 (C-3), 124.6 (C-5), 123.4 (C-4), 123.3 (C-12, 14), 113.3 (C-7)
3-((1-Oxoindolin-2-ylidene)methyl)phenyl 4-methoxybenzoate (A2) light yellow solid, Yield:
45%, mp: 88-90oC, Rf 0.53, IR (ν cm-1) 3373 (N-H) (w); 2988 (=C-H str.) (m); 1745 (C=O)
1
(ester) (s), 1601 (C=O) (amide), 1156 (C-O) (methoxy); H NMR (400 MHz, CDCl3) δ (ppm):

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8.19-8.17 (2H, dd, J12 = 2Hz, J13 = 7Hz, ArNH), 8.14 (1H, s-broad, -NH), 7.81 (1H, s, ArH),
7.75-7.73 (2H11,15, d, J = 8 Hz, ArH), 7.70-7.68 (1H, d, J = 8Hz, ArH) 7.34-7.32 (2H, d, J =
8Hz, ArH), 7.26-7.23 (2H m, ArH), 7.02- 7.00 (2H, dd, J12 = 2Hz, J13 = 7Hz, ArH), 6.91-6.88
13
(1H, m, ArH), 3.91 (3H, s, OCH3); C NMR (100 MHz, CDCl3) 168.65 (C-1), 165.85 (C-16),
163.73 (C-20), 151.37 (C-13), 142.16 (C-8), 134.51 (C-9), 132.43 (C-10), 131.90 (C-18, C-22),
130.38 (C-11, C- 15), 129.85 (C-6), 127.81 (C-3), 122.26 (C-4), 122.09 (C-12, C-14), 120.85 (C-
5), 120.80 (C- 17), 120.76 (C-2), 114.27 (C-19, C-21), 110.01 (C-7), 55.54 (OCH3)
3-((1-Oxoindolin-2-ylidene)methyl)phenyl 3,4-dimethoxybenzoate (A3) light yellow solid,
Yield: 42%, mp: 83-85oC, Rf 0.54, IR (ν cm-1) 3382 (N-H) (w); 2982 (=C-H str.) (m); 1732
1
(C=O) (ester); 1597 (C=O) (amide) (s), 1166 (C-O) (s), 1136 (C-O) (s); H NMR (400 MHz,
CDCl3) δ (ppm): 10.63 (1H, s, ArNH), 7.82-7.80 (3H, d, J=8 Hz), 7.66 (1H, s, ArH), 7.62 (1H, s,
ArH), 7.57-7.55 (1H, d, J=8 Hz, ArH), 7.44-7.42 (2H, d, J= 8 Hz, ArH), 7.27-7.25 (1H, m,
ArH), 7.19-7.17 (1H, d , J=8 Hz, ArH), 6.90-6.86 (2H, m, ArH), 3.89 (3H, s, OCH3), 3.86 (3H,
s, OCH3); 13C NMR (100 MHz, CDCl3) 168.62 (C-1), 164.45 (C-16), 152.98 (C-20), 152.03 (C-
19), 143.72 (C-13), 142.34 (C-8), 137.68 (C-9), 130.23 (C-10), 129.03 (C-11, 15), 126.15 (C-6),
125.95 (C-3), 124.21 (C-4), 123.71 (C-5), 123.31 (C-2), 122.46 (C-22), 122.26 (C-17), 121.82
(C-14, 12), 114.90 (C-7), 107.72 (C-21), 107.35 (C-18), 56.26 (OCH3)
3-((1-Oxoindolin-2-ylidene)methyl)phenyl 3-cyanobenzoate (A4) yellow solid, Yield: 47%,
mp: 155-157, Rf 0.49, IR (ν cm-1) 3361 (N-H) (w); 1469 (-NH bend) (s), 1734 (C=O) (s) (ester),
1601 (C=O) (s) (amide); 1H NMR (400 MHz, CDCl3) δ (ppm): 8.52-8.50 (1H, s, ArH), 8.46-8.43
(2H, m, ArH), 7.71-7.57 (5H, m, ArH), 7.38-7.32 (2H, m, ArH), 7.15-7.12 (1H, m, ArH), 6.96-
13
6.91 (2H, m, ArH); C NMR (100 MHz, CDCl3) 167.52 (C-1), 162.97 (C-16), 151.69 (C-13),
139.85 (C-8), 138.21 (C-9), 136.88 (C-20), 134.23 (C-22), 133.84 (C-18), 132.30 (C-10), 130.81
(C-17), 130.52 (C-21), 129.85 (C-11, 15), 129.10 (C-6), 129.03 (C-3), 122.62 (C-5), 122.13 (C-
4), 122.07 (C-2), 121.90 (C-12, 14), 177.93 (CN), 113.45 (C-7), 112.71 (C-19)
3-((1-Oxoindolin-2-ylidene)methyl)phenyl 3-fluorobenzoate (A5) yellow solid, Yield: 45%,
mp: 161-163, Rf 0.48, IR (ν cm-1) 3372 (N-H) (w); 3067 (=C-H str.) (m); 1637 (C=O) (amide)

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1
(s), 1740 (C=O) (ester) (s), 916 (C-F) (s); H NMR (400 MHz, CDCl3) δ (ppm): 8.75 (1H, s,
ArNH), 8.03-8.01 (1H, d, J=8 Hz, ArH), 7.92-7.90 (1H, m, ArH), 7.81 (1H, s, ArH), 7.76-7.74
(2H, d, J= 8 Hz, ArH), 7.67- 7.65 (1H, d, J=8 Hz, ArH), 7.54-7.50 (1H, m, ArH), 7.39-7.34 (3H,
13
m, ArH), 7.24- 7.22 (1H, m, ArH), 6.94-6.92 (1H, m, ArH), 6.89-6.87 (1H, m, ArH); C NMR
(100 MHz, CDCl3) 168.62 (C-1), 164.45 (C-16), 162.12 (C-19), 152.62 (C-13), 143.72 (C-8),
137.68 (C-9), 131.82 (C-10), 130.57 (C-21), 129.03 (C-11, 15), 126.15 (C-6), 125.98 (C-22),
124.21 (C-5), 123.71 (C-4), 123.31 (C-3), 122.46 (C-14, C-12), 122.26 (C-20, C-7), 121.82 (C-
2), 114.90 (C-18)
3.3 In-vitro biological evaluation
In vitro evaluation of anti-TB potential of the synthesized compounds was done using
Lowenstein Jensen Medium (L. J. Medium) (Canetti, Froman et al. 1963). A stock culture of M.
tuberculosis H37Rv (MTCC–200) strains were procured from Institute of Microbial
Technology, Chandigarh. DMSO was used as diluents / vehicle to get desired concentration of
drugs to test upon Standard bacterial strains. Each synthesized drug was diluted to obtain 2000
µg/ml concentration, as a stock solution and experiment were performed in triplicate. In primary
screening, MIC was determined at 500, 250 and 125 µg/ml and compound A2 showed complete
inhibition at 250 µg/ml, whereas remaining four compounds showed MIC at 125 µg/ml. These
four compounds were further subjected to secondary screening by diluting them to 100 µg/ml,
50 µg/ml, 25 µg/ml, 12.5 µg/ml, 6.250 µg/ml, 3.125 µg/ml and 1.5625 µg/ml concentrations.
After secondary screening out of these four compounds, A1 and A3 were found to possess
comparably better anti-TB activity with the value, 73 µM and 31 µM respectively. The
compounds displayed better activity than the previously reported GyrB inhibitor, novobiocin
having MIC value >200 µM. However the obtained MIC value was found poor in comparison
to fourth generation fluoroquinolone, moxifloxacin (1.26 µM), a GyrA inhibitor (Medapi,
Suryadevara et al. 2015). This disclose that GyrB inhibitor can be further explored to develop
more potent anti-TB agents and oxindole scaffold can be used as lead for designing of GyrB
inhibitors.
<Table 2>
4. Conclusion

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This study is an effort to develop therapeutic agents against resistant TB by a rational designing
of selective, novel and potent GyrB inhibitors. Various oxindole based derivatives were designed
using pharmacophore models generated for GyrB and GyrA domain. The designed compounds
were further validated using structure based techniques, i.e. docking in combination with MD
studies. The designed molecules were synthesized and evaluated for their anti-TB activity by
calculating MIC values using LJ proportion method. Out of five two compounds A1 and A3
having benzoyl and 3,4-dimethoxy benzoyl substitutions showed moderate activity against M.
tuberculosis H37Rv with MIC value of 73 µM and 31 µM. The obtained results lay ground for
further efforts to develop small molecule GyrB domain selective inhibitors for resistant
tuberculosis.
As two compounds A1 and A3 displayed below moderate antitubercular activity as compared to
the standard drug isoniazid and moxifloxacin therefore the designed molecules need further in
silico guided optimization to achieve significant anti-TB potency. Additionally, the molecules
can be further evaluated against resistant anti-TB strain and M. tuberculosis gyrase enzyme using
fluoroquinolones as standard to establish their anti-TB mechanism via inhibition of GyrB
domain.
Conflict of interest: Authors declare no conflict of interest.
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Figure Captions
Fig. 1: Previously generated pharmacophore model of M. tuberculosis GyrB domain mapped
with designed compounds.
Fig. 2: 2D interaction diagram of compound A1 in complex with GyrB domain displaying
important interactions with the active site residues.
Fig. 3: RMSD plot of compound A1 in complex with M. tuberculosis GyrB displaying that the
protein-ligand complex was stable after the MD simulations of 10 ns.
Scheme 1. Synthetic scheme of designed oxindole based analogues.

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Table 1 Five designed compounds with their energies and binding affinity.
-CDOCKER
MM-GBSA binding energy
Compound
Structure
interaction
IDs
GyrBa
GyrAb
-25.72
GyrBc
-76.23
energy (GyrB)
-41.7713
-44.1913
-80.5352
A1
A2
-79.7922
-82.2382
-19.26
-38.59
-74.58
-77.03
OCH3
OCH3
O
O
-46.7133
-44.3761
A3
A4
A5
O
N
H
-77.2543
-58.7274
10.57
-74.99
-60.47
-44.6676
-23.19
a Binding affinity score for GyrB domain in kcal/mol.
b Binding affinity score for GyrA domain in kcal/mol.
c Binding affinity score for GyrB mutant (N499D) in kcal/mol.

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Table 2 In vitro activity data of designed compounds against Mycobacterium tuberculosis
H37Rv using minimum inhibitory assay.
S. No.
Compound code
MIC Mtb H37Rv*
(µM)
A1
A2
A3
A4
A5
73
673
31
1
2
3
136
278
4
5
Standard
Isoniazid
1.4
>200
1.26
Novobiocin(ref 9)
Moxifloxacin(ref 9)
* Minimum inhibitory concentration (MIC) of the compounds on Mtb H37Rv determined
using Lowenstein Jensen medium (conventional method).

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