Structure-based drug design, synthesis and screening of MmaA1 inhibitors as novel anti-TB agents

Article abstract of DOI:10.1007/s11030-020-10107-0

Abstract: Tuberculosis is one of the leading causes of death across the world. The treatment regimens for tuberculosis are well established, but still the control of the disease faces many challenges such as lengthy treatment protocols, drug resistance an

Full text of DOI:10.1007/s11030-020-10107-0

Molecular Diversity
https://doi.org/10.1007/s11030-020-10107-0
ORIGINAL ARTICLE
Structure‑based drug design, synthesis and screening of MmaA1
inhibitors as novel anti‑TB agents
Hymavathi Veeravarapu1,2 · Vasavi Malkhed2 · Kiran Kumar Mustyala2 · Rajender Vadija2 · Ramesh Malikanti2 ·
Uma Vuruputuri2 · Murali Krishna Kumar Muthyala1
Received: 3 April 2020 / Accepted: 15 May 2020
© Springer Nature Switzerland AG 2020
Abstract
Tuberculosis is one of the leading causes of death across the world. The treatment regimens for tuberculosis are well estab-
lished, but still the control of the disease faces many challenges such as lengthy treatment protocols, drug resistance and
toxicity. In the present work, mycolic acid methyl transferase (MmaA1), a protein involved in the maturation of mycolic
acids in the biochemical pathway of the Mycobacterium, was studied for novel drug discovery. The homology model of
the MmaA1 protein was built and validated by using computational techniques. The MmaA1 protein has 286 amino acid
residues consisting of 10 α-helices and 7 β-sheets. The active site of the MmaA1 protein was identifed using CASTp,
SiteMap and PatchDock. Virtual screening studies were performed with two small molecule ligand databases: Asinex syn-
ergy and Diverse_Elite_Gold_Platinum databases having a total of 43,446 molecules and generated 1,30,814 conformers
against the predicted and validated active site of the MmaA1 protein. Binding analysis showed that the residues ASP 19,
PHE 22, TRP 30, TYR 32, TRP 74 and ALA 77 of MmaA1 protein have consistent interactions with the ligands. The hit
ligands were further fltered by in silico ADME properties to eliminate potentially toxic molecules. Of the top 10 molecules,
3-(2-morpholinoacetamido)-N-(1,4-dihydro-4-oxoquinazolin-6-yl) benzamide was synthesised and screened for in vitro anti-
TB activity against Mtb H37Rv using MABA assay. The compound and its intermediates exhibited good in vitro anti-TB
activity which can be taken up for future lead optimisation studies.
Electronic supplementary material The online version of this
article (https://doi.org/10.1007/s11030-020-10107-0) contains
supplementary material, which is available to authorized users.
* Murali Krishna Kumar Muthyala
drmmkau@gmail.com
1
Pharmaceutical Chemistry Research Lab, AU College
of Pharmaceutical Sciences, Andhra University,
Visakhapatnam, Andhra Pradesh 530003, India
2
Molecular Modelling Research Lab, Department
of Chemistry, University College of Science, Osmania
University, Hyderabad, Telangana, India
Vol.:(0123456789)
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Molecular Diversity
Graphical abstract
Structure based virtual screening study was performed using a validated homology model against small molecules from two
virtual compound libraries. Synthesised the lead compound 3-(2-morpholinoacetamido)-N-(1,4-dihydro-4-oxoquinazolin-
6-yl)benzamide obtained from virtual screening. In vitro activity against Mtb H37Rv has given a promising result.
Keywords Tuberculosis · Mycolic acid methyl transferase · MmaA1 · Homology modelling · Virtual screening · MABA
assay
Mycolic acid methyl transferase (MmaA1)
Introduction
Mycobacterium tuberculosis, the causative agent of the
dreadful disease tuberculosis, has an intricate and complex
cell wall structure. The complex, lipid-rich cell envelope
is largely responsible for resistance and virulence of the
mycobacterium which also acts as a permeability barrier.
The cell wall of the mycobacterium is comprised of pepti-
doglycans, arabinogalactans and mycolic acids which are
covalently bonded [9, 10]. Mycolic acids are long-chain
α-alkyl β-hydroxy fatty acids, classifed into alpha, methoxy
and keto-mycolic acids [11, 12]. They are synthesised by
two discrete elongation systems in mycobacteria, type I and
type II fatty acid synthases (FAS I and FAS II, respectively)
[13]. The synthesis of mycolic acids is followed by exten-
sive post-synthetic modifcations and unsaturations. These
Tuberculosis (TB) is an air-borne infectious disease caused
by the bacteria Mycobacterium tuberculosis, one of the top
10 leading causes of mortality across the globe. Accord-
ing to WHO global TB report 2019, it is estimated that 10
million people were afected and 1.2 million died due to
this dreadful disease [1]. The main course of treatment for
TB is the directly observed treatment, short-course (DOTS)
therapy [2]. Currently, there are many new drugs in the dis-
covery pipeline and some of them have been approved for
the treatment like Bedaquiline (2012), Delamanid (2013)
and Pretomanid (2019) [3–5]. The main drawbacks with TB
treatment are resistance, long duration of treatment and drug
toxicity. Therefore, this supports the search for new drug
[6–8].
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Molecular Diversity
Fig. 1 Biochemical pathway of
MmaA1 protein showing the
role of MmaA1 protein in the
biosynthesis of mycolic acids
post-synthetic modifcations are brought about by a family of
S-adenosyl-l-methionine (SAM)-dependent enzymes, which
use meromycolic acid as a substrate to generate cis- and
trans-cyclopropanes and other mycolic acids [14]. Mycolic
acid methyl transferases belong to the mycolic acid cyclo-
propane synthases family, which play an important role in
the synthesis of methoxy mycolic acids and have been des-
ignated as MmaA1–4 to indicate their involvement in the
methoxy mycolic acid biosynthesis [15].
There is no reported 3-D structure for MmaA1 protein
available in the protein data bank, and hence, we initiated
homology modelling to facilitate the virtual screening study.
Materials and methods
Software
MmaA1 protein is essential for the synthesis of trans-
and keto-mycolic acids in the mycobacterium, shown in
Fig. 1. The MmaA1 protein converts cis-olefn oxygenated
mycolate precursor to a trans-olefn with an adjacent methyl
branch, i.e. to a trans-mycolic acid. Increase in trans-mycolic
acid leads to increased cell wall rigidity, increased drug
resistance and altered colony morphology [16]. Therefore,
MmaA1 also acts as a branch point in the synthesis of trans-
and keto-mycolic acids. Thus, inhibiting MmaA1 protein
leads to inhibition of synthesis of mycolic acids resulting
in loss of cell wall rigidity and ultimately cell death. There-
fore, targeting MmaA1 protein can result in potential drug
candidates for TB treatment [17, 18].
Webservers used for Active site prediction: CASTp,
PatchDock.
Active site prediction: SiteMap, Schrodinger, LLC, New
York, NY, 2010.
Docking: Glide, version 5.6, Schrodinger, LLC, New
York, NY, 2010.
Docking analysis: Accelrys Discovery Studio, Schrod-
inger Suite.
ADMET studies: QikProp, version 3.4, Schrodinger,
LLC, New York, NY, 2011.
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Molecular Diversity
structures by comparing them with the 3-D profles of cor-
rect protein structures having high scores. ProSA is a widely
used tool to check potential errors in 3-D models of proteins
which are obtained either from experimental elucidation,
from protein engineering or theoretical models. The pro-
gram gives a z-score plot and energy plot for the protein.
The energy of the structure is evaluated using a distance-
based and solvent exposure potential of protein residues. The
z-score is indicative of overall model quality of the protein
3-D structure. The ProSA energy plot determines the local
quality of the 3-D model by plotting energy as a function of
amino acid sequence position. Positive values correspond to
erroneous parts, whereas negative values indicate a stable
and good model.
Homology modelling of MmaA1 protein
The FASTA sequence of the protein was retrieved from the
ExPASy Proteomic server (Uniprot ID: P9WPB1) [19]. The
sequence was then screened for similarity search against all
the proteins deposited in Protein Data Bank [20] using tem-
plate and similarity search programs such as Basic Local
Alignment Search Tool (BLASTp) [21] and JPred3 server
[22]. The alignment of identifed template and target proteins
was executed using ClustalW [23]. The 3-D model of the
protein was generated using MODELLER 9v13 [24].
Model validation
The obtained 3-D model of the protein was evaluated using
PROCHECK [25] and Verify_3D [26] available from the
Structural Analysis and Verifcation Server (SAVES) and
ProSA server [27]. The PROCHECK server analyses the
stereochemical quality of the protein and gives an assess-
ment of overall quality of the structure compared to well-
refned structures of similar resolution, which is given by the
Ramachandran (RC) plot [28, 29].
Active site prediction
The binding of the ligand in the active site of the protein is
brought about by the microenvironment, which is created by
folds of the 3-D structure of protein into cavities or pockets.
The knowledge of structure of the cavities of proteins helps
in designing novel ligands, enzyme inhibitors, understanding
their binding modes and protein dynamics. In the present
study, active site of the protein was identifed using CASTp
The accuracy of a protein 3-D structure can be deter-
mined by Verify_3D, which tests the quality of protein
Scheme 1 Synthesis scheme of 3-(2-morpholinoacetamido)-N-(3, 4-dihydro-4-oxoquinazolin-7-yl)benzamide
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Molecular Diversity
(Computed Atlas of Surface Topography of proteins) server
[30], SiteMap [31] and PatchDock online webserver [32].
PatchDock was used to carry out protein–ligand docking of
the MmaA1 protein with its natural substrate S-adenosyl-
N-decyl-aminoethyl (SADAE) [33]. A grid was generated
by the Receptor grid generation module of Glide taking into
consideration the predicted active site residues.
score and glide energy were used for prioritising the ligand
molecules.
In silico ADME prediction
The absorption, distribution, metabolism and elimination
are crucial properties for the clinical success of a drug can-
didate. Nearly 50% of the drugs fail because of poor ADME
properties [41, 42]. Advances have been made in the in silico
prediction of ADME properties of molecules, which resulted
in a number of programs available for predicting several
parameters/descriptors for this purpose. Predicting ADME
reduces animal testing, reduces cost and time and provides
reliable toxicity prediction. In the present study, the ADME
properties were predicted using QikProp [43].
Virtual screening
Virtual screening is a fast and cost-efective tool for screen-
ing of compound databases in search of novel lead mol-
ecules. This method can be used for screening large librar-
ies of chemicals for compounds that complement targets of
known structure [34]. Computational screening or docking
methods are improving at an incredible rate providing newer
versions which have become complementary alternatives to
high-throughput screening [35]. In the present work, Grid-
based Ligand Docking with Energetics (Glide) module of
Schrodinger Suite [36] was used for docking. The virtual
screening process is carried out in four stages in Glide,
namely protein preparation, ligand preparation, grid gen-
eration and virtual screening.
Experimental
All the solvents and chemicals were purchased from com-
mercial sources and used without further purifcation. The
reactions were monitored on pre-coated silica gel TLC
using appropriate mobile phase. Visualisation of the spots
was done with the help of UV at 254 nm. Biotage Initia-
tor system was used for microwave reactions. For column
chromatography, stationary phase: silica gel, mobile phase:
chloroform–methanol, gradient elution method was used.
Melting points were noted by using DigiMelt (Stanford
Research Systems, USA) and are uncorrected. IR spectral
data were recorded on Bruker ALPHA-T FTIR system using
KBr pellet method. 1H and 13C NMR spectra were recorded
on Bruker (400 MHz for 1H and 100 MHz for 13C) instru-
ment. CDCl3 and DMSO-d6 were used as NMR solvents and
tetramethylsilane (TMS) as an internal standard. Chemical
shifts (δ) values were referenced to the residual solvent peak
and reported in ppm, and all coupling constant (J) values
were given in Hz. The following multiplicity abbreviations
are used: (s) singlet, (d) doublet, (t) triplet, (m) multiplet,
(q) quartet, (dd) double doublet. HRMS data were measured
on Agilent 6530 Q-TOF LC-HRMS system, ESI method,
positive mode. The instrument was tuned using an Agilent
tune mix. A reference solution (m/z 121.0509, m/z 922.0098)
was used to correct small mass drifts during the acquisition.
Analyses indicated by the symbols of the elements or func-
tions were within 0.4% of theoretical values.
The MmaA1 protein was prepared for docking by submit-
ting it to the Protein preparation wizard module of Schrod-
inger Suite. OPLS-2005 force feld was used for allocation of
bond orders [37]. This step is followed by energy minimisa-
tion using Impref module of Schrodinger Suite, using OPLS-
2005 force feld, with a cutof RMSD of 0.3 Ǻ between suc-
cessive conformations, in the minimisation process [38].
The 3-D coordinates of the ligands were optimised using
LigPrep module of Schrodinger Suite [39]. The stereoi-
somers with specifc chiralities were retained to generate
fve conformers per ligand. The pre-fltering process also
includes elimination of reactive functional groups and to
improve the pharmacokinetic properties of the ligands for
further study. The Receptor Grid generation module in Pro-
tein preparation wizard was used for grid generation.
In the virtual screening stage, the ligand molecules are
docked at the predicted active site of the protein using Glide
module of Schrodinger suite. Glide uses a series of modules
which act as hierarchical flters, namely high-throughput vir-
tual screening, standard precision (sp) and extra precision
(XP) to minimise false positives and false negatives from
entering into fnal phases of docking results [40]. The glide
Fig. 2 Conserved domains of
MmaA1 protein
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Molecular Diversity
Table 1 Template search results
S. no.
Name of the server Parameters considered for template selection E value
PDB code
of template
protein
for MmaA1 protein
1
2
BLAST
JPred3
Sequence position specifcity
4e−116 1KP9_A
1e−89 1KP9_A
Secondary structure prediction, solvent acces-
sibility and coiled-coil region prediction
The servers predicted 1KP9_A as template for the MmaA1 protein
The synthesis of compound (3) was brought by a series
of reactions using isatin as the starting material. Isatin
(0.068 mmol) was dissolved in 12 ml sulphuric acid at 0 °C.
To this sodium nitrate (0.068 mmol) dissolved in sulphuric
acid was added dropwise and kept for stirring for 4 h at 0 °C
[45]. The reaction mixture was poured onto crushed ice, fl-
tered and dried to obtain a yellow colour solid, 5-nitro isatin.
The 5-nitro isatin (0.99 mmol) was subjected to oxidation
at RT for 15 min using 5% sodium hydroxide solution and
hydrogen peroxide (0.032 mmol). Then, dil. HCl was added
and pH adjusted to 1 to obtain 5-nitro anthranilic acid [46].
It was then fltered and dried to obtain a pale yellow solid.
To 5-nitro anthranilic acid (5 mmol) and formamide
(50 mmol), catalytic amount of acetic acid was added and
kept in microwave oven for 10 min at 300 W [47]. The reac-
tion mixture was quenched on ice and fltered to obtain a
brown colour solid, 7-nitroquinazolin-4(3H)-one. To 7-nit-
roquinazolin-4(3H)-one (2.61 mmol), in ethanol as solvent,
ammonium chloride (26.16 mmol) dissolved in water and
iron powder (10.39 mmol) were added and kept for refux for
4 h to obtain the compound (3), 7-aminoquinazolin-4(3H)-
one. The reaction mixture was fltered under vacuum. The
fltrate was evaporated under vacuum to obtain a grey col-
our solid which was further re-crystallised using methanol/
charcoal to obtain grey coloured shiny needle shape crystals.
The synthesis of the title compound (4),
3-(2-morpholinoacetamido)-N-(3,4-dihydro-4-oxoquina-
Synthesis
of 3‑(2‑morpholinoacetamido)‑N‑(3,4‑dihydro‑4‑ox‑
oquinazolin‑7‑yl) benzamide
All the top 10 compounds obtained from virtual screen-
ing study were analysed for novelty and synthetic feasibil-
ity based on literature search and SciFinder search studies.
The compound 3-(2-morpholinoacetamido)-N-(3,4-dihydro-
4-oxoquinazolin-7-yl) benzamide was selected for synthesis
based on this analysis. The scheme of synthesis for the title
compound is given in Scheme 1. The starting materials for
synthesis in this scheme are 3-aminobenzoic acid and isatin.
The compound (1), 3-(2-chloroacetamido) benzoic acid was
synthesised from 3-aminobenzoic acid (0.4 mmol) and chlo-
roacetyl chloride (0.5 mmol) in dry benzene at RT by stirring
for 5 h [44]. After completion of the reaction as indicated
by TLC, the reaction mixture was poured onto crushed ice,
stirred, fltered and dried under vacuum to obtain a white
colour solid, which was further re-crystallised from chloro-
form/methanol (9:1) mixture. The compound (2), 3-(2-mor-
pholinoacetamido) benzoic acid was obtained by treating
compound (1) (2.34 mmol) and morpholine (5.68 mmol)
with ethyl acetate as solvent and heating at 80 °C for 2 h. A
brown colour gummy solid was obtained which was separated
and purifed using chromatographic column (dry packing
method), silica gel as stationary phase and chloroform–metha-
nol mobile phase eluted by gradient method.
Fig. 3 Sequence alignment of MmaA1 protein with the template (1KP9_A) showing identical residues in blue colour, strongly similar residues
in dark blue colour and weakly similar residues in yellow colour
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Molecular Diversity
Fig. 4 The secondary structure
of MmaA1 protein shown in
purple colour helices and sheets
predicted by PDBsum. The red
line represents β-hairpin motif
Table 2 Details of secondary
structure of the MmaA1 protein
showing α-helices
S. no.
Start of α-helix
End of α-helix
No. of resi- Sequence
dues
1
2
3
4
5
6
7
8
9
ASP19
LEU44
GLY76
ARG96
TYR146
MET185
GLU207
GLU228
ARG246
GLU255
ALA23
ASP57
5
14
10
11
11
13
11
17
7
DDFFA
LEEAQLAKVDLALD
GALVRAVEKY
RNHYERSKDRL
YLTFFERSYDI
MSDLRFLKFLRES
EPDIVDNAQAA
QQHYARTLDAWAANLQA
RERAIAV
TYR85
ALA106
ILE156
SER197
ALA217
ALA244
VAL252
ARG274
10
20
EEVYNNFMHYLTGCAERFRR
The secondary structure of MmaA1 protein has 10 α-helices predicted using ProFunc of PDBsum
zolin-7-yl) benzamide was brought about by acid amide
coupling [48]. To 3-(2-morpholinoacetamido) benzoic acid,
(1.403 mmol, 1 eq), DIEA (1.665 mmol, 2.5 eq) was added
under ice cold conditions and kept for stirring for 5 min, then
EDC HCl (1.987 mmol, 1.2 eq), HOBt (2.383 mmol, 1.2 eq)
and 7-aminoquinazolin-4(3H)-one (2.383 mmol, 0.26 eq)
were added and kept for stirring at RT for 72 h. The solvent
was evaporated under vacuum, and re-crystallised with hex-
ane–ethyl acetate (1:2) to obtain a highly hygroscopic brown
colour solid.
In vitro anti‑TB activity study of 3‑(2‑morph
olinoacetamido)‑N‑(3,4‑dihydro‑4‑oxoquin
azolin‑5‑yl)benzamide and its intermediates
Microplate Alamar Blue Assay (MABA) is a rapid, high-
throughput, inexpensive dye-based cell viability assay which
uses Alamar blue as indicator for anti-mycobacterial drug
screening. MABA helps in quantitative determination of drug
susceptibility of replicating M. tuberculosis [49, 50]. The
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Molecular Diversity
Table 3 Details of the secondary structure of the MmaA1 protein
showing β-sheets
S. no. Start of β-sheet End of β-sheet No. of
residues
Sequence
1
2
3
4
5
6
7
THR66
ASN88
ALA115
ARG131
ARG162
THR220
ILE277
VAL70
LEU92
ARG118
PHE135
THR170
LEU226
THR285
5
5
4
5
9
7
9
TLLDV
NVIGL
AEAR
RIVSF
RMLLHSLFT
TIEHVQL
INVAQFTMT
The secondary structure of the MmaA1 protein has 7 β-sheets pre-
dicted using ProFunc of PDBsum
Fig. 6 Ramachandran plot of MmaA1 protein
Table 4 Ramachandran plot statistics of MmaA1 protein
Description
No. of
amino
acids
Percentage
Residues in most favoured regions
Residues in additional allowed regions
Residues in generously allowed regions
Residues in disallowed regions
243
19
0
0
262
92.7%
7.3%
0.0%
0.0%
100.0%
Number of non-glycine and non-proline
residues
Number of end-residues (excl. Gly and Pro)
2
15
Fig. 5 3-D model of MmaA1 protein
Number of glycine residues (shown as
triangles)
Number of proline residues
Total number of residues
7
286
synthesised compound 3-(2-morpholinoacetamido)-N-(3,4-
dihydro-4-oxoquinazolin-5-yl)benzamide and its intermedi-
ates were evaluated for their in vitro anti-tubercular activity
against H37Rv strain using the Microplate Alamar Blue sus-
ceptibility test, and the activity is given as minimum inhibi-
tory concentration (MIC) in μg/ml.
Fig. 2. The predicted domain S-adenosyl methionine binding
site extends from ASP 70–LEU 170. The template search
result from BLAST predicted the Apo-form of Mycolic acid
cyclopropane synthase (Cmaa1), PDB code: 1KP9_A to
have the highest identity of 57% and E value of 4e−116 and
JPred3 server predicted an E value of 1e−89 for the protein
1KP9_A. The E values with the corresponding templates
are given in Table 1.
Results and discussion
Structural evaluation of MmaA1 protein
The FASTA sequence of MmaA1 protein (Accession no.
P9WPB1) was obtained from Expasy Proteomics tool (Uni-
prot) from Server Expert Protein Analysis System (Expasy
SWISS-PROT/TrEMBL) database. The conserved domains
of the protein predicted using BLAST server are shown in
The MmaA1 protein sequence alignment with the tem-
plate protein 1KP9_A shows a 76% sequence similarity
shown in Fig. 3. Therefore, the protein Mycolic acid cyclo-
propane synthase (Cmaa1) was taken as the template for
model generation based on the sequence alignment and
1 3

Molecular Diversity
Fig. 7 Verify_3D plot of MmaA1 protein
Fig. 8 z-plot of MmaA1 protein obtained ProSA webserver. The
Fig. 9 ProSA energy plot of MmaA1 protein
black spot encircled corresponds to MmaA1 protein
predicted E values. The secondary structure of MmaA1 pro-
tein is shown in Fig. 4 which consists of ten α-helices and
seven β-sheets. The residues corresponding to the secondary
structure which are predicted using ProFunc (Prediction of
protein function from 3-D structure) [51] of PDBsum web-
server are given in Tables 2 and 3.
β-hairpins consist of two β-strands which are anti-parallel
and are hydrogen bonded together.
Model refnement and validation
The α-helices in the secondary structure of MmaA1 pro-
tein form an embellishment pattern shown in Fig. 5 which
is characteristic of Mycolic acid S-adenosyl methyl trans-
ferases (SAM-MTs). The protein core consists of seven
β-strands parallel to each other except β-strand 7, fanked
on each side by three helices. There is a β-hairpin motif
between the seven residues in β strand 6 (THR 220–LEU
226) to the nine residues in β-strand 7 (ILE 277–THR 285).
The Ramachandran plot of MmaA1 protein is shown in
Fig. 6 obtained from SAVES server. The plot statistics are
given in Table 4. The plot statistics show that 92.7% of
amino acid residues are in most favoured region, 7.3% in
additionally allowed region and none of the residues in the
generously allowed or disallowed regions. Therefore, the
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Molecular Diversity
Table 5 Active site regions predicted for MmaA1 protein
S. no.
Server
Active site residues
1
2
CASTp
SiteMap
TYR32-CYS34, ASP69-ARG80, LEU92-ARG101, GLN120-GLU123, SER134-ALA140
SER13-PHE22, TRP30, TYR32, GLU46, ASP69-ALA81, LEU92-LEU 94, HIS98,
TRP122, SER134-TYR146, LEU165, ASN260-TYR264
3
PatchDock
TYR 32, THR 33, TRP 74
generated 3-D model is a good-quality model in terms of
stereochemical quality.
Table 5. Figure 10 depicts the active site of MmaA1 pro-
tein generated using PyMOL [52]. The MmaA1 protein
predicted active site extends in the residue domain ranging
from SER13–PHE22, TRP30, TYR32–CYS34, GLU46,
ASP69–ALA81, LEU92–ARG101, GLN120–GLU123,
SER134–TYR146, LEU165, ASN 260–TYR264. The
active site of MmaA1 protein was generated and consid-
ered for grid generation in virtual screening using these
predicted residues.
The Verify_3D plot of MmaA1 protein is shown in Fig. 7.
The plot shows that 86.7% of the residues have an averaged
3D-1D score≥0.2. A good-quality structure requires at least
80% of the amino acids to have a score≥0.2 in the 3D/1D
profle.
The MmaA1 protein was submitted to ProSA webserver,
and a z-score of − 6.69 was obtained. The plot shows
z-scores of all protein chains in PDB determined by X-ray
crystallography (light blue colour region) and by NMR spec-
troscopy (dark blue region) shown in Fig. 8.
Virtual screening
Figure 9 shows the energy profle of MmaA1 protein
obtained using ProSA web server. The obtained ProSA
energy plot for the model shows maximum residues within
negative region of energy, which indicate that the MmaA1
model is a stable model.
In the present study, structure-based virtual screening was
carried out for MmaA1 protein using two small molecule
ligand databases: Asinex synergy and Diverse_Elite_Gold_
Platinum using protocols reported earlier [53, 54]. Asinex
synergy database consisting of 36,411 compounds was sub-
jected to ligand preparation. The stereoisomers with spe-
cifc chiralities were retained to generate fve conformers
per ligand. The stable conformers were retained, and an
output of 1,17,159 stereoisomeric structures were gener-
ated. The generated conformers were subjected to HTVS
mode to obtain an output of 12,896 structures. These output
structures were further screened successively in SP and XP
modes with a fltration ratio of 10% at each stage of dock-
ing as reported earlier [55]. This screening process gave an
output of 128 structures of docked complexes. The criteria
for fltration of ligands at each stage include good docking
and scoring poses.
Active site identifcation
The binding site predictions for MmaA1 protein from
CASTp, SiteMap and protein ligand docking are given in
The protocol followed for Asinex synergy database was
applied to the virtual screening of Diverse_Elite_Gold_
Platinum database as well. The Diverse_Elite_Gold_
Platinum database consists of 7035 molecules and after
subjecting to ligand preparation gave an output of 13,655
conformers and further virtual screening process through
HTVS, SP and XP modes gave an output of 13 docked
complexes. All these complexes can be considered for the
identifcation of potent ligands as inhibitors for MmaA1
protein. A total of 141 docked complexes were obtained
from virtual screening process with Glide scores ranging
from − 13 to − 12. The resulting docked complexes were
visually inspected, analysed and prioritised based on Glide
score. A sample of top 10 molecules (M1–M10) are given
in Table 6 prioritised based on Glide docking score. The
Fig. 10 Active site of MmaA1 protein. The protein is represented as
green colour ribbon model, and active site residues are represented as
red colour spheres
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Molecular Diversity
Table 6 Ligands showing Glide score and Glide energy with intermolecular interactions and bond distance in Angstroms
S. no.
Structure
Glide score
Glide energy
Intermolecular interactions
Bond
distance
(Å)
1
−13.4
−53.5
Hydrogen bonds
ALA77:H-M1:O19
M1:H28-TRP30:O
Pi–Pi interactions
TYR32-M1:N48
PHE22-M1:N48
Hydrogen bonds
2.038
2.126
2-methyl-1-(2-(4-methyl-3-oxo-3,4-dihydro-2H-benzo[b][1, 4]oxazin-6-yl carba-
moyl)benzyl)-1H-imidazol-3-ium
4.695
4.715
2
−12.7
−65.20
TRP74:HE1-M2:N2
GLY76:M2:N-O16
M2:H29-TRP30:O
M2:H34-ASP19:OD1
M2:H52-ASP19:OD1
Pi–Pi interactions
PHE22-M2:N55
1.950
2.386
1.804
2.271
2.088
3-(2-morpholinoacetamido)-N-(3,4-dihydro-4-oxoquinazolin-7-yl) benzamide
4.924
6.752
M2:NZ-LYS51
3
−12.4
−56.17
Hydrogen bonds
TRP74:HE1-M3:N2
M3:H29-TRP30:O
M3:H58-ASP16:OD1
Pi–Pi interactions
PHE22-M3:N55
1.950
2.386
1.804
(S)-3-((4-6-oxo-2-(1-phenyl ethylamino)-3,6-dihydropyrimidin-4-yl)piperidinyl)
methyl)pyridinium
4.924
4
5
−12.4
−51.1
Hydrogen bonds
TRP74:HE1-M4:N1
M4:H29-TRP30:O
Pi–Pi interactions
PHE22-M4:N55
Hydrogen bonds
1.800
2.142
3-((4-(2-(4-methylbenzylamino)-6-oxo-3,6-dihydropyrimidin-4-yl)piperidinium-
1-yl)methyl) pyridinium
5.804
−12.2
−61.9
M5:H33-TRP30:O
M5:H35-ASP19:OD1
Pi–Pi interactions
M5-LYS51:NZ
2.094
1.939
4-(2-oxo-2-(3-(4-oxo-1,4-dihydro quinazolin-6-yl carbamoyl) phenyl amino)ethyl)
morpholin-4-ium
6.050
6.966
M5-LYS51:NZ
1 3

Molecular Diversity
Table 6 (continued)
S. no.
Structure
Glide score
−11.8
Glide energy
−45.6
Intermolecular interactions
Bond
distance
(Å)
6
Hydrogen bonds
ALA77:H-M6:O8
M6:H32-TRP30: O
Pi–Pi interactions
PHE22-M6
1.946
1.884
(S)-3-((3-((2,4-dimethyl-3-oxo-3,4-dihydro-2H-benzo[b][1, 4]oxazin-6-ylamino)
methyl)phenoxy)methyl)pyridinium
4.047
6.520
TYR32-M6:N52
7
−11.6
−49.0
Hydrogen bonds
TYR32:H-M7:N9
TRP74:HE1-M7:N2
M7:H31-TRP30:O
M7:H33-GLY71:O
Hydrogen bonds
2.484
2.001
1.636
2.266
(R)-4-(2-(6-(3-isopropyl-1H-pyrazol-5-yl)-2-methylpyrimidin -4-ylamino)-2-phe-
nylethyl) morpholin-4-ium
8
−11.3
−54.4
TYR32:H-M8:O20
TRP74:HE1-M8:N15
M8:H36-TRP30:O
Pi–Pi interactions
M8-PHE135
1.907
2.025
1.687
(S)-N-(2-(2-cyclopropyl-6-oxo-3,6-dihydropyrimidin-4-yl)-1-(4-fuorophenyl)
ethyl)-1H-pyrazole-5-carboxamide
5.463
9
−11.3
−47.8
Hydrogen bonds
ALA77:H-M9:O18
M9:H23-TRP30:O
M9:H24-GLY71:O
Pi–Pi interactions
M9-VAL31:HG22
PHE22-M9:N38
TYR32-M9:N38
Hydrogen bonds
2.323
2.058
2.255
3-(3-((4-methoxybenzamido)methyl)-1H-1,2,4-triazol-5-yl)pyridinium
2.907
4.316
5.511
10
−11.3
−49.4
TRP74:HE1-M10:N1
M10:H32-TRP30:O
Pi–Pi interactions
PHE22-M10
2.913
2.195
(R)-2-(1-(4-chloro-1H-pyrazol-1-yl)propan-2-yl)-6-(4-fuoro benzyl amino)
pyridine-4(1H)-one
4.776
Ligand structures and intermolecular interactions between the protein MmaA1 and the ligands of the docked complexes. The functional groups
forming hydrogen bonds and pi–pi interactions have been highlighted in circles
1 3

Molecular Diversity
Fig. 11 a Binding interaction of 3-(2-morpholinoacetamido)-N-(3,4-dihydro-4-oxoquinazolin-7-yl) benzamide with MmaA1 protein, b 2-D view
of binding interaction of 3-(2-morpholinoacetamido)-N-(3,4-dihydro-4-oxoquinazolin-7-yl) benzamide with MmaA1 protein
Table 7 Predicted ADME or pharmacokinetic values of the top ten docked molecules for MmaA1 protein
S.no. Mol.wt. CNS Donor HB Accept HB Rule of fve Rule of three QPlogPo/w QPlog BB QPlog HERG %Human oral
absorption
M1
M2
M3
M4
M5
M6
M7
M8
M9
M10
436
407
389
389
407
389
406
367
309
361
1
−2
1
0
−2
0
1.0
3.0
2.0
2.0
3.0
1.0
2.0
3.0
2.0
2.0
5.75
12.2
7.5
7.5
12.2
7.0
7.2
7.0
7.25
5.5
1
0
0
0
0
0
0
0
0
0
2
0
0
0
0
1
0
0
0
0
5.06
0.47
3.26
2.96
0.39
4.11
3.58
2.56
1.76
3.87
−0.29
−1.36
−0.64
−0.75
−1.34
−0.53
−0.31
−1.35
−0.93
−0.59
−6.69
−7.16
−7.43
−7.12
−7.08
−6.51
−6.42
−5.46
−5.87
−5.90
100
58
86
82
57
100
93
83
87
100
1
−2
−1
0
binding pose, hydrogen bond and pi–pi interaction of the
synthesised ligand, 3-(2-morpholinoacetamido)-N-(3,4-
dihydro-4-oxoquinazolin-7-yl) benzamide with MmaA1
protein from docked complex is shown in Fig. 11a and
b. The protein ligand interactions were visualised using
Accelrys Discovery Visualiser [56].
such as molecular weight and descriptor values of donor
hydrogen bonds, acceptor hydrogen bonds are in accept-
able range. All the top 10 ranking molecules obey Lipin-
ski’s rule of fve, Jorgensen rule of three and various other
important pharmacokinetic parameters. The molecules
M1, M6 and M10 show 100% human oral absorption,
and the molecules M3, M4, M7, M8 and M9 show more
than 80% human oral absorption. The other important
descriptors which are responsible for the drug candidate
to be successful in the drug discovery are CNS activitiy,
QPlogBB (predicted brain/blood partition coefcient) and
QPlogHERG (predicted IC50 value for blockage of HERG
K + channel) and are in permissible limits for the top 10
molecules in the present study. Therefore, the molecules
ADME analysis
The evaluation of ADME properties is an essential part
of drug discovery to minimise failures of drug candidates
in clinical phase. In the present study, the ADME proper-
ties were predicted using QikProp module of Schrodinger
suite, given in Table 7. The physicochemical properties
1 3

Molecular Diversity
obtained from virtual screening in the present study have
ADME properties within acceptable range which show
that these molecules have good drug-like properties.
Compound (4):
3‑(2‑morpholinoacetamido)‑N‑(3,4‑dihydro‑4‑oxo‑
quinazolin‑5‑yl) benzamide
Highly hygroscopic solid, Yield: 50%; IR (KBR): 1109,
1
1444, 1607, 1663, 2860, 2919 cm−1; HNMR (400 MHz,
Characterisation of synthesised compounds
Compound (1): 3‑(2‑chloroacetamido) benzoic acid
White solid; Yield: 93%; mp: 220.6 °C; IR (KBr): 1593,
DMSO-D6): 1.25 (d, J = 6.4 Hz, 5H), 1.99 (s, 2H), 2.70
(s, 1H), 3.14 (s, 3H), 7.09 (d, J = 7.6 Hz, 1H), 7.40 (m,
3H), 7.52 (d, J = 8.4 Hz, 1H), 7.70 (d, J = 8 Hz, 1H),
7.76 (d, J=6.4 Hz, 1H), 7.85 (d, J=8.4 Hz, 1H), 9.90 (s,
1H); 13CNMR (100 MHz, DMSO-D6): δ 169.30, 168.88,
143.34, 139.15, 136.43, 129.29, 128.13, 125.89, 124.05,
122.27, 120.95, 119.11, 110.64, 66.55, 62.47, 60.24, 53.81,
21.56, 17.93, 14.54, 12.98; HRMS (ESI): m/z calcd. for
C21H21N5O4 [M + H]+: 408.16, found: 408.1672; Anal
calcd. for C21H21N5O4: C: 65.01; H: 5.46; N: 13.78; found
C: 65.00; H: 5.44; N: 13.76%; anti-TB activity MIC (H37Rv
assay): 100 μg/ml.
1
1675, 3088, 3272 cm−1; HNMR (400 MHz, DMSO-D6):
δ 4.27 (s, 2H), 7.48 (t, J=8, 1H), 7.68 (d, J=7.6 Hz, 1H),
7.82 (t, J = 8, 1H), 8.23 (s, 1H), 10.51 (s, 1H); 13CNMR
(100 MHz, DMSO-D6): δ 43.99, 120.54, 123.93, 125.10,
129.65, 131.88, 139.16, 165.38, 167.49; HRMS (ESI): m/z
calcd. for C9H8ClNO3 [M+H]+: 214.02, found 214.0276;
Anal calcd. for C9H8ClNO3: C: 50.60; H: 3.77; N: 6.56%;
found C: 50.58; H: 3.75; N: 6.55%; anti-TB activity MIC
(H37Rv assay): 25 μg/ml.
Compound (2): 3‑(2‑morpholinoacetamido) benzoic
acid
Conclusion
In the present study, we studied MmaA1, a protein involved
in the maturation of mycolic acids needed for cell wall syn-
thesis of Mycobacterium tuberculosis, using structure-based
drug design techniques. The 3-D model of the protein was
built using Modeller and validated using Ramachandran
plot, Verify_3D and ProSA. Active site of the MmaA1 pro-
tein was identifed using CASTp, SiteMap and protein ligand
docking. Virtual screening was performed using Schrodinger
suite, and 141 docked complexes were obtained. The top
10 molecules prioritised based on Glide docking score are
reported in this study. The residues ASP 19, TRP 30, TYR
32, GLY 71, TRP 74, GLY 76, ALA 77 and PHE 135 of the
MmaA1 protein have shown important binding interactions
with the ligands. ADME properties of the molecules were
calculated using QikProp module of Schrodinger suite. All
the top 10 molecules obey Lipinski’s rule of fve, Jorgensen
rule of three and various other important pharmacokinetic
parameters. In the present study, we have reported the syn-
thesis of 3-(2-morpholinoacetamido)-N-(3,4-dihydro-4-ox-
oquinazolin-7-yl) benzamide and its intermediates. The syn-
thesised compound and its intermediate compounds have
shown good micromolar anti-tubercular activity. These
results along with acceptable ADME properties suggest
this class of compounds may furnish candidates for future
development of novel anti-TB drugs.
Pale brown solid; Yield: 90%; mp: 91.5 °C; IR (KBr): 1106,
1517, 1662, 2854, 2923 cm−1; 1HNMR (400 MHz, DMSO-
D6): 2.88 (s, 1H), 3.14 (s, 3H), 3.65 (t, J=9.2 Hz, 6H), 7.43
(t, J=8 Hz, 1H), 7.64 (d, J=8 Hz, 1H), 7.86 (d, J=8 Hz,
1H), 8.25 (s, 1H), 9.92 (s, 1H); 13CNMR (100 MHz, DMSO-
D6):δ 169.61, 168.65, 138.63, 136.86, 128.55, 124.75,
122.29, 120.97, 66.55, 65.31, 62.47, 53.61, 44.07; HRMS
(ESI): m/z calcd. for C13H16N2O4 [M+H]+: 265.11, found:
265.1164; Anal calcd. for C13H16N2O4: C: 59.08; H: 6.10;
N: 10.60%; found C: 59.06; H: 6.10; N: 10.58%; anti-TB
activity MIC (H37Rv assay): 25 μg/ml.
Compound (3): 7‑aminoquinazolin‑4(3H)‑one
Grey solid; Yield: 80%; mp: 232.90C; IR (KBr): 1676, 2908,
2951, 3170 cm−1; 1HNMR (400 MHz, DMSO-D6): δ 5.6 (s,
2H), 7.08 (t, J=8.8 Hz, 1H), 7.18 (d, J=2.4 Hz, 1H), 7.38
(d, J=8.8 Hz, 1H), 7.75 (d, J=2.8 Hz, 1H), 11.8 (s, 1H);
1HNMR-D2O exchange (400 MHz, DMSO-D6): δ7.13 (d,
J = 8 Hz, 1H), 7.17 (s, 1H), 7.41 (d, J= 7.6 Hz, 1H), 7.78
(s,1H); 13CNMR (100 MHz, DMSO-D6): δ 106.58, 122.61,
124.11, 128.52, 140.10, 140.75, 148.34, 161.13; HRMS
(ESI): m/z calcd. for C8H7N3O [M + H]+: 162.06, found:
162.0662; Anal calcd. for C8H7N3O: C: 59.62; H: 4.38; N:
26.07%; found C: 59.60; H: 4.36; N: 26.05%; anti-TB activ-
ity MIC (H37Rv assay): 100 μg/ml.
Acknowledgements The author HV acknowledges Department of Sci-
ence and Technology (DST), Government of India, for fnancial sup-
port under the Women Scientist Scheme (WOS-A) vide reference no.
SR/WOS-A/CS-1031/2014. The authors HV, VM, KKM, RV, RM and
UV acknowledge the Principal and Head, Department of Chemistry,
University College of Sciences, Osmania University, Hyderabad, India,
1 3

Molecular Diversity
15. Takayama K, Wang C, Besra GS (2005) Pathway to synthesis
and processing of mycolic acids in mycobacterium tuberculo-
sis. Clin Microbiol Rev 18(1):81–101. https://doi.org/10.1128/
cmr.18.1.81-101.2005
for providing the facilities to carry out this work. The authors HV
and MMK acknowledge the Principal, AU College of Pharmaceutical
Sciences, Andhra University, Visakhapatnam, India, for providing the
facilities to carry out this work.
16. Yuan Y, Crane DC, Musser JM, Sreevatsan S, Barry CE III
(1997) MMAS-1, the branch point between cis and trans cyclo-
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Compliance with ethical standards
Conflict of interest The authors declare that they have no confict of
interest.
17. Brossier F, Veziris N, Trufot-Pernot C, Jarlier V, Sougakof W
(2011) Molecular investigation of resistance to the anti tubercu-
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