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Combinatorial Chemistry & High Throughput Screening, 2013, 16, 000-000 1

1386-2073/13 $58.00+.00 2013 Bentham Science Publishers
High Throughput Screening of 7-Methylpicene-1,2-Diol as Arylamine N-
Acetyltransferase (NAT) Inhibitor to Establish a Isoniazid Supplement in
Anti-Tubercular Therapy
Abhishek Chowdhury
*,1
, Paulomi Paul
2
and Manabendra Dutta Choudhury
1
1
Bioinformatics Centre (DBT-BIF),
2
Department of Biotechnology, Assam University Silchar, Assam 788011, India
Abstract: Mycobacterium tuberculosis (Mtb), due to its unusual organization crosses different immune barriers and
causes tuberculosis. The advent of multidrug resistance tuberculosis (MDR-TB) has attained alarming situation. Hence,
computational drug design has been performed in this work to find potent molecules for this purpose.
Isoniazid is a widely used frontline drug against tuberculosis. But reports justified the inactivity of isoniazid on
acetylation by Arylamine N-acetyltransferase (NAT). 35 countries were highlighted to have isoniazid resistance from
survey in 1998. Hence, Mtb NAT has been selected as the target in the present case and hundred compounds were
screened in order to find potent NAT inhibitor to raise the efficacy of isoniazid.
Molecular docking with Biosolveit LeadIT and Autodock 4.2 simulation was performed. The result showed 7-
methylpicene-1, 2-diol to have -26.77 and 8.26 kcal/mol score in LeadIT and Autodock 4.2. The work validated 7-
methylpicene-1, 2-diol to be a potent NAT inhibitor to supplement isoniazid.
Keywords: Anti-tubercular drug, Arylamine N-acetyltransferase, isoniazid, MDR-TB, NAT, 7-methylpicene-1, 2-diol.
INTRODUCTION
Due to the alarming increase in Multidrug resistance
tuberculosis (MDR-TB) cases, the need of new compound to
encumber the growth of tuberculosis has become a necessity
[1]. According to the report of Times of India on 13 October
2011, the multidrug resistance tuberculosis (MDR-TB) has
attained 3.3% of new tuberculosis cases. Emergence of 0.44
million new cases and death of 0.15 million have also been
reported in 2009. There is also report of more than 2.8
million cases of MDR-TB infection and average annual
death reached to 9 million [2]. The global predominance of
multidrug-resistant strains clearly indicates that the old
targets and old drugs for Mycobacterium tuberculosis are no
longer effective. Compounds with higher efficacy against
well-established targets of widespread biological importance
have proved to be effective for the discovery of new
tubercular antidotes.
Isoniazid has been included as a frontline drug in the
combination therapy relying on the treatment regimen of
tuberculosis [3]. Isoniazid prodrug gets metabolized by the
action of catalase-peroxidase enzyme to formactive drug [4].
The Isoniazid moiety then disrupts the mycolic acid
biosynthesis to stop mycobacterial cell wall maintenance [5-8].
Modified Isoniazid gets covalently attached to NAD
+
and
inhibits mycolic acid synthesis [8]. kasA encoded -ketoacyl
ACP synthase also signifies an alternative target, inhibited by
the formation of a covalent ternary complex of Isoniazid [9].
Arylamine N-acetyltransferase (NAT) is cytosolic enzymes,
which help in the acetylation using acetyl-CoA as the acetyl


*Address correspondence to this author at the Bioinformatics Centre (DBT-
BIF), Assam University Silchar, Assam 788011, India; Tel: +91-8822 210
211; Fax: 03842-270920; E-mail: abhishek@bioinfoaus.ac.in
group donor [10]. Many hydrazine- and arylamine-based drugs
are substrates for NAT. Therefore, Arylamine N-
acetyltransferases is a proven target for antitubercular drug
design and hence, is considered the target in this work.
Though, modification of the target by the pathogen itself
especially in MDR-TB cannot be overlooked, to find a potent
NAT inhibitor, one hundred compounds were screened based
on their property of hitting cellular proteins and containing free
phenolic groups. Those compounds need to be validated with
the drug-like properties and their non-toxic nature. The NAT is
a well-established target and hence the present work tries to find
a potent inhibitor of NAT by the computational approach.
Piceno[13,14-b]oxirene-1,2-diol is reported to have
mutagenic activity that forms DNA adduct in cells of
eukaryotes by inducing 6-thioguanine-resistant mutants in
Chinese hamster cells. It has also been seen that the OH group
in 9th position is responsible for the chemical reactivity of this
compound [11]. Hence, new derivatives were prepared using
this compound with side chain modification to remove toxic
components but keeping the OH group at 9th position to
retain the efficacy.
The hypothesis of the present work lies in that if a
compound can block NAT with better efficacy than Isoniazid; it
can prevent the acetylation of Isoniazid and hence can raise the
Isoniazid efficacy to stop MDR-TB. Hence, the present work
was designed to find a potent Isoniazid supplement against
multi drug resistance tuberculosis.
MATERIALS AND METHODS
Target Selection
Arylamine N-acetyltransferases (NATs) of Mycobacterium
tuberculosis was selected as the target (contains 283 residues)
2 Combinatorial Chemistry & High Throughput Screening, 2013, Vol. 16, No. 9 Chowdhury et al.
and the 3D structure was obtained fromthe homology modeling
taking Arylamine N-acetyltransferases (NATs) of
Mycobacterium smegmatis (PDB ID: 1W6F) (contains 278
residues) as a template using Modeller v9.10 [12]. Theactive
site residues were screened out in Q-site finder [13].
Ligands
Piceno[13,14-b]oxirene-1,2-diol was found to be partially
mutagenic and also was reported to have better chemical
reactivity against prokaryotes since it can form adduct with
DNA/protein. Considering this, combinatorial library of
hundred compounds were generated using SmiLib v2.0 [14]
substituting the free epoxy group and leaving the OH groups
in its original positions to retain its efficacy. The compounds
generated were converted to .sdf format using OpenBabel
[15] and used for docking.
Toxicity and Drug Likeness
Mobyle@rpbs online portal [16,17] was used for
ADME/Tox screening of the selected ligands and the results
were recorded. Actelion (OSIRIS) property explorer [18]
was used to screen the drug likeness.
Molecular Docking
Molecular docking was performed using Biosolveit
LeadIT [19] and Autodock 4.2 [20] with five completely
non-toxic derivatives separately against the target. Docking
results i.e., docking energy, docked amino acid residues,
hydrogen bond, and bond energy were recorded using
LeadIT while binding energy and interacting surface area
using Autodock 4.2 in Table 1.
Comparison with Isoniazid
Isoniazid has been taken from Pubchem (CID 3767) and
was docked with NAT both in LeadIT and Autodock 4.2 to
compare its efficacy against the target in order to validate
whether the presence of the designed ligands will help
Isoniazid in its activity or not.
RESULTS
The modelled NAT has 283 amino acid residues.
Different models were generated using homology modelling
technique and the model with more stability and accuracy
was selected. Ramachandran plot [21] was generated for the
accepted model i.e., Model-1 (Figs. 1, 2).
Out of the hundred combinatorial library derivatives 7-
methylpicene-1,2-diol (Ligand1) proved complete non-toxicity
and high oral absorption by passing ADME/Tox filter and
Actelion property explorer (Fig. 3). There was no sign of
mutagenecity for the Ligand1 in Actelion.
Molecular docking with the target revealed that Ligand1 has
higher affinity of -26.37 Kcal/mol than that of Isoniazid (-17.79
Kcal/mol). The binding energy with Autodock 4.2 was recorded
to be -8.26 and -4.50 Kcal/mol for Ligand1 and Isoniazid
respectively (Table 1, Figs. 4, 5). The figures were generated by
LeadIT [19] and Dockingserver [22].
DISCUSSION
Mycobacterium smagmatis was found to have optimum
phylogenetic distance with M. tuberculosis than the other
strains, which allows both variation and homology to persist
[23]. For this very reason, NAT enzyme of M. smegmatis was
used as the homology modeling template.
Since the protein target was has very few amino acid
residues, the Q-site finder showed active sites which were
composed of TYR69, TYR195, VAL95, THR109, HIS110,
GLN 135 mainly. The Ligand1 showed its binding affinity
towards the THR109 and HIS110 residues which were among
the core residues of active site. This showed the efficacy of
Ligand1 to block the target.
The comparison also showed that Isoniazid has one
common residue in bonding i.e., THR109 (Fig. 6) which may
confer that Ligand1 has better potential in targeting the active
sites than Isoniazid. In an earlier work, it has been reported that
katG helps in catalase-peroxidase activity and hence turn the
bacterium resistant to Isoniazid [9].
Though enoyl carrier protein (ACP) reductase is the target
for Isoniazid but NAT acetylates isoniazid pro-drug and hence
reduces its activity [4]. Hence, to find a new way in TB
treatment a NAT was chosen as the target.
The docking study is based on the principle that least
docking score shows more binding stability [24]. The Ligand1
showed least score than Isoniazid hence it could have better
binding with the target, which could block the target. The
Autodock 4.2 results also showed the same and authenticate the
findings [22].
Since the comparison showed better results for Ligand1 than
Isoniazid in both LeadIT and Autodock 4.2, it can be concluded
that the compound Ligand1 will work better to supplement
Isoniazid in treating tuberculosis (especially MDR-TB).


Table 1. Different Parameters in the Comparison of Drug Efficacy of 7-Methylpicene-1,2-Diol and Isoniazid in LeadIT and
Autodock 4.2

LeadIT Autodock 4.2
Compound
Score
(Kcal/mol)
Bonded
Residue
Bond Length
()
Bond Energy
(Kcal/mol)
Binding Energy
(Kcal/mol)
Interaction Surface
(cm
2
)
7-methylpicene-1,2-diol -26.37
H36-HIS110A
H37-THR109A
1.89
2.09
-4.70
-4.61
-8.26 663.253
Isoniazid -17.79
O1-CYS70A
H5-THR109A
1.82
1.99
-4.70
-4.70
-4.50 433.699
High Throughput Screening of 7-Methylpicene-1,2-Diol Combinatorial Chemistry & High Throughput Screening, 2013, Vol. 16, No. 9 3




















Fig. (1). Ramachandran plot of the modeled Arylamine N-
acetyltransferase protein (Complete).
The least score of isoniazid also prove less affinity
towards NAT and hence ligand1 is likely to interact with
NAT in competitive manner and hence isoniazid may escape
this catalysis process. In a similar work with kas-A which is
analogous to the target of isoniazid, two compounds with
similar functional entities to that of the screened compound
were screened for their anti-tubercular efficacy based on
LeadIT score good binding efficacy were predicted which
was followed for the present study as a template work [25].





















Fig. (2). Ramachandran plot of the modeled Arylamine N-
acetyltransferase protein (Residue specific).
CONCLUSION
Taking all the above into consideration, the compound
i.e., 7-methylpicene-1,2-diol was suggested to have anti-
















Fig. (3). Actelion property view of 7-methylpicene-1,2-diol showing non-toxicity and drug like properties.
4 Combinatorial Chemistry & High Throughput Screening, 2013, Vol. 16, No. 9 Chowdhury et al.
tubercular potential against NAT and can also supplement
Isoniazid to eradicate multidrug-, mono drug- and
extensively drug- resistant pathotype of Mycobacterium
tuberculosis. The compound may need further in vivo and in
vitro study to justify its drug conflicts with Isoniazid if any.
This prediction if preceded further may lead a renaissance in
the era of tubercular drug design using computational tools.













Fig. (4). LeadIT Docking pattern comparison (2D) of 7-methylpicene-1,2-diol (left) and Isoniazid (right).












Fig. (5). Autodock 4.2 binding pattern comparison of 7-methylpicene-1,2-diol (left) and Isoniazid (right) showing similarity in binding
pattern.











Fig. (6). Comparison of Protein interaction for Isoniazid (left) and 7-methylpicene-1, 2-diol (right) showing comparative common binding
mode and better efficacy of 7-methylpicene-1, 2-diol than Isoniazid.
High Throughput Screening of 7-Methylpicene-1,2-Diol Combinatorial Chemistry & High Throughput Screening, 2013, Vol. 16, No. 9 5
CONFLICT OF INTEREST
The authors confirm that this article content has no
conflict of interest.
ACKNOWLEDGEMENTS
Authors acknowledge the help of DBT Govt. of India for
establishing the Bioinformatics Centre in Assam University,
Silchar where the work has been carried out and the
DeLCON facility for literature search.
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Received: January 12, 2013 Revised: May 16, 2013 Accepted: May 21, 2013