Identification of novel scaffold using ligand and structure based approach targeting shikimate kinase

Article abstract of DOI:10.1016/j.bioorg.2020.104083

Tuberculosis (TB) remains a major global health problem. It causes ill-health among millions of people each year and rank as the second leading cause of death from an infectious disease worldwide, after the human immunodeficiency virus (HIV). Shikimate kinase is one of the major enzymes targeted for TB. Most approaches to overcome TB were based on synthesis and screening of a known compounds to obtain a few representatives with desired potency. In this study, we have applied a virtual screening approach which combines ligand- and structure-based approaches to screen a large library of compounds as a starting point for the identification of new scaffolds for the development of shikimate kinase inhibitors. The combined approach has identified 2 new scaffolds as potential inhibitors of shikimate kinase. To prove the approach, few of the molecules and their derivatives, a total of 17 compounds, were synthesized. The compounds were tested for biological activity and shows moderate activity against shikimate kinase. The shikimate kinase enzyme inhibition study reveals that the compounds showed inhibition (IC₅₀) at concentrations of 50 μg/mL (Compounds 21, 22, 24, 25, 26, 27, 30, 32, 34) and 25 μg/mL (14, 19, 23, 31, 33).

Full text of DOI:10.1016/j.bioorg.2020.104083

BioorganicChemistry102(2020)104083
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Identification of novel scaffold using ligand and structure based approach
targeting shikimate kinase
⁎
M.B. Rahul Reddya, Sivakumar Kullampalayam Krishnasamyb, M.K. Kathiravana,c,
a Department of Pharmaceutical Chemistry, SRM College of Pharmacy, SRM IST Kattankulathur, Kancheepuram, Tamil Nadu 603203, India
b Department of Pharmaceutical Chemistry, KMCH College of Pharmacy, Kovai Estate, Kalapatti Road, Coimbatore, Tamil Nadu 641048, India
c 209, Dr APJ Abdul Kalam Research Lab, SRM College of Pharmacy, SRM IST Kattankulathur, Kancheepuram, Tamil Nadu 603203, India
A R T I C L E I N F O
A B S T R A C T
Keywords:
Tuberculosis (TB) remains a major global health problem. It causes ill-health among millions of people each year
and rank as the second leading cause of death from an infectious disease worldwide, after the human im-
munodeficiency virus (HIV). Shikimate kinase is one of the major enzymes targeted for TB. Most approaches to
overcome TB were based on synthesis and screening of a known compounds to obtain a few representatives with
desired potency. In this study, we have applied a virtual screening approach which combines ligand- and
structure-based approaches to screen a large library of compounds as a starting point for the identification of
new scaffolds for the development of shikimate kinase inhibitors. The combined approach has identified 2 new
scaffolds as potential inhibitors of shikimate kinase. To prove the approach, few of the molecules and their
derivatives, a total of 17 compounds, were synthesized. The compounds were tested for biological activity and
shows moderate activity against shikimate kinase. The shikimate kinase enzyme inhibition study reveals that the
compounds showed inhibition (IC50) at concentrations of 50 µg/mL (Compounds 21, 22, 24, 25, 26, 27, 30, 32,
34) and 25 µg/mL (14, 19, 23, 31, 33).
Tuberculosis
Shikimate kinase inhibitors
Pyrazole derivatives
1. Introduction
wide in terms of estimated cases per year. The recommended standard
chemotherapeutic regimen for TB treatment is prescribed under Di-
Tuberculosis (TB) remains a global health concern with about 10
million cases in 2018 [1]. TB remains the top infectious killer world-
wide. Although some countries are significantly accelerating their TB
response, most World Health Organization (WHO) regions and many
high-burden countries are still not on track to reach the 2020 goal of the
End TB strategy. TB is a communicable disease that is a major cause of
ill health, also one of the top 10 causes for death worldwide and the
leading cause of death for a single infectious agent ranking above
Human Immuno Virus (HIV). It is caused by the bacillus Mycobacterium
tuberculosis [2], which is spread when people who are sick with TB
expel bacteria into the air.
rectly Observed Treatment Short-course (DOTS), lasting for minimum
6 months [3].
Currently there are 23 drugs for the treatment of drug-susceptible
TB, multidrug-resistant TB are in phase I, II and III trials. These drugs
consist of 13 new compounds, three other drugs (Bedaquiline,
Delamanid and Pretomanid) that have already got the regulatory ap-
proval and 7 repurposed drugs [4]. FDA approved TB drug Bedaquiline
(Sirturo) for the treatment of Multi Drug Resistant – TB (MDR-TB) [5,6]
Sirturo precisely inhibits mycobacterial adenosin 5-triphosphate syn-
thase, an enzyme that is essential for the final step in ATP production by
oxidative phosphorylation [7]. Its related risk of potentially lethal heart
problems has emphasized the unmet and urgent need for the develop-
ment of safer antitubercular drugs with new targets and new mechan-
isms of action to treat resistant forms of the TB disease.
There are an estimated 1.2 million TB deaths among HIV-negative
people in 2018 and an additional 251,000 deaths among HIV-positive
people. Globally 7.0 million new cases of TB were notified in 2018 [1],
an increase from 6.4 million notified 2017. Most of the increase in
global notifications of TB cases since 2013 is explained by trends in
India and Indonesia, the two countries that rank first and third world
One promising mechanism for antitubercular agent is the inhibition
of shikimate kinase (SK) in the shikimate pathway [8]. The shikimate
pathway is used in a variety of bacteria, including Mycobacterium
Abbreviations: TB, Tuberculosis; DOTS, Directly Observed Treatment Short-course; MDR-TB, Multi Drug Resistant Tuberculosis; MtSK, Mycobacterium tuberculosis
shikimate kinase; MACCS, Molecular access system; MOE, Molecular operating environment
⁎ Corresponding author at: Department of Pharmaceutical Chemistry, SRM College of Pharmacy, SRM IST Kattankulathur, Kancheepuram, Tamil Nadu 603203,
India.
https://doi.org/10.1016/j.bioorg.2020.104083
Received 1 April 2020; Received in revised form 4 July 2020; Accepted 6 July 2020
Availableonline15July2020
0045-2068/©2020ElsevierInc.Allrightsreserved.

M.B. Rahul Reddy, et al.
BioorganicChemistry102(2020)104083
Fig. 1. List of shikimate kinase inhibitors reported in the literature.
tuberculosis, for the production of chorismite, a precursor for aromatic
amino acids [9] and other aromatic compounds [10]. Mammals do not
have the shikimate pathway enzymes necessary for de novo synthesis of
these amino acids but rather obtain them from the diet [10]. Therefore,
inhibitors of SK are anticipated to be discriminating antitubercular
drugs. Shikimate kinase is the one of the seven enzymes involved in the
shikimate pathway.
1.1. List of shikimate kinase inhibitors
To identify new scaffolds as inhibitors of shikimate kinase, we se-
lected two computational approaches the ligand-based screening
method, similarity search and the structure-based approach, molecular
docking. The ligand-based screening approach is based on the hy-
pothesis that molecules with similar structure possibly have similar
activity. Thus, for the initial screening and understanding of current
available scaffolds, we collected the shikimate kinase inhibitors which
were reported in the literature. Then the similar structures are screened
using the crystal structure of shikimate kinase for identifying the po-
tential interaction required for the scaffold to act as inhibitors of shi-
kimate kinase. As a first step, the list of potent shikimate kinase in-
hibitors reported in the literature is shown in Fig. 1 [15–20].
Shikimate Kinase in Mycobacterium tuberculosis (MtSK), is the fifth
enzyme in the pathway, catalyzes the phosphorylation of shikimate
(SA) using ATP as a phosphoryl donor to form shikimate 3-phosphate
(S3P) and ADP [11]. With no mammalian counterpart, MtSK represents
a favorable target for the design of drugs specific to the M. tuberculosis
pathogen with reduced risk of toxicity in the human host [12,13].
Shikimate kinase is encoded by aroK and is essential for the survival of
Mycobacterium tuberculosis [14].
2

M.B. Rahul Reddy, et al.
BioorganicChemistry102(2020)104083
Scheme 1. Procedure for synthesis of Compound 14.
2. Materials and methods
query molecule and the way these groups are linked together. Each
node in the tree is labelled with a set of features representing chemical
properties of the part of the molecule corresponding to the node. The
comparison of feature trees is based on matching subtrees of two fea-
ture trees onto each other. Unlike fingerprint-based similarity search,
the minimum FTrees similarity score between the query and the target
molecules called as similarity threshold was set to a fixed value as 0.8.
The output of FTrees visual similarities is a particular similarity score
for each query-target pair.
2.1. Compound dataset
Enamine database is a commercial supplier of chemicals with more
than 15 million small molecules which are categorized into building
blocks, fragments and screening compounds (www.enamine.net). They
have introduced one of the largest databases which contains more than
720 million synthetic accessible drug-like molecules called as Readily
Accessible (REAL) database. The compounds in the database are syn-
thesizable using one-pot synthesis protocols. The REAL database is
further classified into drug-like, lead-like, fragments, covalent modi-
fiers, chemical classes and natural product-like compounds. For our hit-
identification, we have downloaded REAL Diverse drug-like database
with over 15 million diverse compounds. The compounds in the data-
base comply with “rule of 5” and Veber criteria with molecular weight
maximum of 500, SlogP contains less than 5, Hydrogen bond acceptors
and donors with less than or equal to 10 and 5, respectively and
TPSA ≤ 140. In addition, the database is with the absence of PAINS and
toxic compounds.
2.3. Molecular docking
The docking studies were performed using MOE-Dock module
available in Molecular Operating Environment (MOE) 2018.01 [24].
The molecular docking approach is a two-stage process with pose
generation and scoring the complexes. For the pose generation, we have
selected the triangle matcher algorithm which was aligned using the
ligand triplets on the alpha sphere triplets of the protein. Then the
generated poses were scored on the interaction with amino acids in the
binding site of the protein using two rescoring functions, London dG
and GBVI/WSA dG.
2.2. Similarity search
For our docking studies, we have selected the crystal structure of
shikimate kinase in complex with ADP and shikimate (2IYQ.pdb) was
downloaded from Protein Data Bank (PDB) [14]. Firstly, the enzyme
was prepared by the following steps, removing water molecules, adding
hydrogen atoms, assigning the partial charges to all atoms and energy
minimization by adopting the default parameters in each module im-
plemented in MOE 2018.01. The co-crystallized ligand shikimate was
used to define the binding site for docking the ligands. Secondly, the
accuracy and suitability of MOE for the present system was validated by
The molecules similar to the selected query molecule are screened
against the downloaded Enamine dataset using FTrees similarity search
from BiosolveIT GmbH FTrees (http://www.biosolveit.de/FTrees)
[21,22]. FTrees-based approach is a complex feature tree instead of
linear fingerprint representations such as MACCS [23] or PubChem
fingerprints (http://pubchem.ncbi.nlm.nih.gov). Each of the feature
trees represents hydrophobic fragments and functional groups of the
3

M.B. Rahul Reddy, et al.
BioorganicChemistry102(2020)104083
Scheme 2. Procedure for synthesis of Compounds 19–34.
reproducing the conformation of shikimate which resulted in a RMSD
value of 0.43 Å. Finally, the selected hit molecules identified from
virtual screening were docked into the binding site of shikimate kinase.
The top 10 docked poses for each ligand was selected and analyzed the
interactions with shikimate kinase using 2D ligand interaction diagram
and visual inspection.
substituted pyrazole derivatives (19 – 22) were methylated using me-
thyl chloride to yield compounds 31 – 34.
A total of seventeen derivatives of the selected compounds from the
library were synthesized using Schemes 1 and 2.
The structured of the synthesised compounds were characterized by
spectral analysis (IR, NMR and Mass). IR spectra of compound 14 de-
monstrated disappearance of primary amine (N-H2) peak present in the
intermediate compound 13, and appearance of amide (CON-H) char-
acteristic absorption band at 3430 and 1698 cm−1 respectively.
Compound 14 showed characteristic absorption band of pyrazolone
cyclic amide (CON-H) at 1705 cm−1; characteristic of arylchloride
moiety (Ar-Cl) absorption band at 1089 cm−1. Compounds 20, 24, 28,
& 32 showed appearance of NO2 (AreCeNeO) characteristic stretching
bands at 1550–1475 cm−1. Compounds 21, 25, 29, & 33 exhibited aryl
Chemical synthesis
The selected identified compounds were synthesized using the
above Scheme 1. The pyrazole (9) ring is synthesized by reacting
ethylacetoacetate and hydrazine. The Pyrazole (9) was oxidized to form
the corresponding acid derivative (10). In another process, 2,6-di-
chloroaniline is nitrated and then further coupled with chloro(methyl-
sulfonyl)methane, which on reduction yielded compound 13. Pyrazole
acid (10) and Compound 13 were combined using amide bond in
presence of DCI to form the final product (Compound 14).
halide (Ar-C-F) characteristic stretching bands at 1069 – 1073 cm−1
.
Some more derivatives of the selected compounds were synthesized
using Scheme 2. Substituted hydrazines and 3-oxobutanenitrile were
cyclised to form the pyrazole (15). Pyrazole 15 was oxidised in pre-
sence of sodium hydroxide to form the carboxylic acid derivative of the
pyrazole (16), which on treatment with thionyl chloride yielded the
acid chloride derivative (17). This compound 17 reacted with methyl
amine forming compound 18. Compound 18 on reaction with sub-
stituted benzoyl chlorides yielded the derivatives 19 – 30. The non-
The second series of synthesized pyrazole compounds 19 – 34 were
confirmed by the presence of –NH/-N, and –N]C pyrazole ring system
stretching bands at 3505 – 3675, 1652 – 1694 cm−1 respectively.
Synthesized final compounds 19 – 34 demonstrated disappearance of
primary amine (N-H2) from the intermediate compound 18, and ap-
pearance of amide (CON-H) characteristic absorption band at 3430, and
1705–1698 cm−1 respectively. Proton assignments in 1H NMR spectra
for the titled pyrazole compounds 19 – 34 showed signal at δ 12.10 to
4

M.B. Rahul Reddy, et al.
BioorganicChemistry102(2020)104083
3. Results and discussion
3.1. Similarity search
In similarity searching, compounds with the known biological ac-
tivity are utilized as reference to search against the molecules in a
database. The fingerprint representations calculated for the query and
database molecules are compared in a pair-wise manner to identify
novel scaffolds as inhibitors of shikimate kinase. Recently Timo and his
coworkers reported the inhibitors targeting M. Tuberculosis identified
using in silico approaches from ligand- and structure-based drug design
methodologies [26]. Among the reported molecules, one of the pyr-
azolone derivatives is the potent inhibitors of shikimate kinase (Fig. 2)
is selected for our virtual screening approach. This lead pharmacophore
combines the features of the available shikimate kinase inhibitors
mentioned in Fig. 1 i.e., the hydrazide, amide, and cyclic structure with
two nitrogen (molecule 1, 5, 6). Briefly, the pyrazolone derivative was
identified by screening the molecules that are designed on the basis of
combination of pharmacophore features from hydrazone and amide
pharmacophore features which is known to inhibit shikimate kinase
efficiently [20] (Fig. 2).
Thus, in our study, one of the most potent pyrazolone derivatives 8
(Fig. 2) is used as the query molecule. The selected query molecule has
to be searched against the molecules from Enamine database. This
database is one of the largest suppliers of molecules which provide high
quality of purchasable compounds with a size of 43.6 million. The
MACCS fingerprint was calculated for the molecules in the database and
the search using the query molecule 8. The top 1000 molecules iden-
tified from the similarity search using MOE-similarity was utilized for
the molecular docking studies.
3.2. Structure analysis and docking studies
Fig. 2. Development of novel scaffold pyrazolone derivatives using the com-
bined pharmacophore features from known hydrazone and amide derivatives
which acts as shikimate kinase inhibitors.
Currently in RCSB Protein Data Bank (PDB) [27] a total of 49 crystal
structures have been reported for shikimate kinase. The structures of
shikimate kinase have been complexed with nucleotides include ATP
and ADP, the endogenous ligand shikimate and its product shikimate 3-
phosphate, inhibitors and also available in different states explaining
their mechanism of action in detail. The shikimate kinase enzyme
transfers the phosphate group from ATP to shikimate and forms as
shikimate 3-phosphate. The phosphate group transfer mechanism and
the conformation movement of the enzyme can be understood clearly
from the crystal structures in apo and complex states. The shikimate
kinase consists of three domains including SB (shikimate binding do-
main), a core domain containing highly conserved phosphate binding
loop (P‐loop), and the “lid”, a highly flexible domain in open and closed
conformation [28,29,30]. The reported structures were analyzed in
detail and the complex with ADP and shikimate (PDB ID: 2IYQ) was
selected for the docking study because the conformations of SB domain
in open and closed states of the enzyme were similar [6]. Among these
three domains in shikimate kinase, the SB is responsible for the transfer
mechanism of phosphate group and the substrate analogs have shown
inhibition of shikimate pathway. Shikimate is found to be stabilized
within the binding site through hydrogen bond interactions with the
amino acid residues D34, R58, G80, and R136. Furthermore, residues
P11, I45, F49, F57, E61, G79, G81, P118, and L119 contributed within
to form the binding site for shikimate [14] (Fig. 3). The docking studies
of the reference compound 8 shows that it occupy the same binding site
as the shikimate and form interaction with D34, R58, G81, and R117,
The selected crystal structure and the docking approach were vali-
dated by redocking studies. The shikimate structure obtained from the
crystal structure was redocked and found to have a top rank pose with
an RMSD value of 0.43 Å. During the docking simulations, the entire
amino acid residues in the enzyme were considered rigid. This confirms
the selected docking approach and the scoring function is appropriate
for the structure and the binding site.
12.16 (s, 1H, NH, D2O exchangeable) and carboxamide (d, 1H, CONH-
CH3) demonstrated characteristic signals at δ 7.56 – 7.60. All the syn-
thesized compounds 19 – 22 & 31 – 34 showed signal at δ 7.0 – 7.45 (m,
5H, Ar-H), while compounds 23 – 30 showed peak at 6.9 – 7.45 (m, 4H,
Ar-H). All the synthesized compounds 19 – 34 showed characteristic
peak of methylene (–CH2-) and methyl (N-CH3) signal at δ 3.96 – 4.10 &
2.96 – 3.05 respectively. On the other hand, Compounds 19, 23, 27 and
31 showed additional typical aromatic methyl signal at δ 2.56 – 2.60.
Compounds 31 – 34 showed the characteristic peaks of methyl group in
pyrazole ring at δ 3.86. Furthermore, the titled compounds were con-
firmed by mass spectra (m/z values).
2.4. Biological testing
The shikimate kinase enzyme inhibition studies were performed
using the method described by Simithy et al. [25], with a slight mod-
ification. Test compounds at different concentrations viz., 100, 50, 25,
12.5, 6.25 µg/mL, and 15 nM of shikimate kinase were preincubated for
15 min in a micro-centrifuge tube containing 455 mL of assay buffer
(100 mM Tris-HCl pH 7.5, 50 mM KCl, and 5 mM MgCl2) at 25 °C. The
reaction was initiated by the addition of an aqueous solution of shi-
kimic acid and ATP (2 and 0.2 mM, final concentrations, respectively)
and quenched after 5 min by the addition of 2 mL of 100% formic acid.
The total volume of the reaction mixture was 500 mL. The presence of
shikimate-3-phosphate after incubation indicates enzyme activity. The
shikimate-3-phosphate in the final solution was determined by using
HPLC. Inhibitory concentration is the conc. at which the compounds
inhibit the enzyme and the absence or decrease in the amount of shi-
kimate-3-phosphate in the final solution.
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M.B. Rahul Reddy, et al.
BioorganicChemistry102(2020)104083
Fig. 3. (A) The crystal structure of shikimate
kinase along with ADP (colored green) and shi-
kimate (colored blue) are shown in stick re-
presentation. The crystal structure of shikimate
kinase is represented in ribbon and colored in
cyan. (B) The comparison of docked pose (co-
lored blue) and crystal structure (colored yellow)
of shikimate. (C) The putative docked pose of the
reference compound, 8 and (D) 2D interaction
diagram of 8 with the residues in the binding
pocket of shikimate kinase. Oxygen atoms are
colored in red, nitrogen atoms in blue, hydrogen
in silver white, and phosphor atoms in orange.
(For interpretation of the references to colour in
this figure legend, the reader is referred to the
web version of this article.)
The hit molecules identified from similarity search was docked into
the binding pocket (SB) of the shikimate kinase. The process was di-
vided further into two step process. In the first step, the entire 5000
molecules identified from similarity search were docked using MOE-
Dock by considering the binding site of the enzyme as rigid. In the
second step process, the top 1000 compounds identified from rigid
docking were subjected to an additional docking step with flexible
binding of the enzyme.
was analyzed in detail by visual inspection. On the basis of interaction
and we selected 10 molecules found interesting and possibly show in-
hibition against the shikimate kinase. The 2D interaction of the 10 se-
lected molecules with the shikimate kinase is provided in Fig. 4 (A-J).
On the basis of the docking results from query molecules 8 and the
amino acids in the binding pocket we search for compounds forming
key electrostatic interacting residues include Lys15, Asp32, As34,
Arg58 and Arg136. Thus, in our docking results we explored for the
ligands interacting with the specified important amino acid residues
need to interact with the hit molecules. The assumed hypothesis is that
the molecules forming interaction similar to the selected query mole-
cules and possibly form similar potency obtained as Compound 8. The
selected ten compounds are pyrazole amides comprising of hydrogen
bond acceptors, one aromatic ring, electron rich halogens and the
aromatic ring substituted with carbonyl (C]O) or sulphonyl (O]
S = O) groups. Possibly the selected ten molecules could be synthesized
in our lab. The selected ten molecules (I-X) were shown in Fig. 5.
The top 5000 molecules obtained from similarity search were pro-
cessed and energy minimized in MOE and docked by considering the
binding site as rigid. Then the resulted binding poses obtained for
molecules from rigid docking were analyzed. The energy values of the
molecules were found in the range from −8.22 to −5.31 kcal/mol. On
the basis of the docking score the top 1000 molecules were selected and
subjected to flexible docking. In this step, the binding site of the enzyme
was considered flexible. During the docking simulation the side chains
of the amino acids were flexible and allow the ligand molecules to dock
deeper into the binding pocket. The energy values of the resulted ligand
poses were found to be in the range from −9.50 to −6.63 kcal/mol. In
the docking process, the energy values of the ligand molecules were
improved due to the flexibility of the binding pocket. From the docking
results of flexible binding site and ligand, the top 100 diverse molecules
were analyzed. The interaction pattern of these 100 diverse molecules
3.3. Chemistry
From the selected 10 compounds the compounds which are possible
to synthesize in our lab were explored. Among the 10 compounds, III
and X were selected for synthesis along with some of their derivatives
6

M.B. Rahul Reddy, et al.
BioorganicChemistry102(2020)104083
Fig. 4. The selected ten molecules from the virtual screening and their 2D interaction diagram specifying the interaction of the molecules with the binding pocket of
shikimate kinase. Their docking score obtained from the flexible docking are also provided.
7

M.B. Rahul Reddy, et al.
BioorganicChemistry102(2020)104083
G)
H)
VII, Escore = -8.01
VIII, Escore = -7.40
I)
J)
X, Escore = -7.52
IX, Escore = -7.95
Fig. 4. (continued)
(total 17 compounds, 14, 19 to 34). The selected identified compounds
and their analogs were synthesized using Schemes 1 and 2 as described
in the materials and methods section and tested for their enzyme in-
hibition study. The reaction progress and purity of the synthesized
compounds were monitored by thin layer chromatography (TLC). The
compounds were synthesized in good yields with high purity (Table 1).
Structures of the synthesized compounds were established on the basis
of spectral data (IR, 1H NMR, and mass).
earlier by Simithy et al. [16], with slight modification. Test compounds
were tested at different concentrations viz., 100, 50, 25, 12.5, 6.25 µg/
mL, and 15 nM of shikimate kinase. The shikimate kinase enzyme in-
hibition study reveals that the compounds showed inhibition (IC50) at
concentrations of 50 µg/mL (Compounds 21, 22, 24, 25, 26, 27, 30,
32, 34) and 25 µg/mL (14, 19, 23, 31, 33). The molecules identified
from ligand- and structure-based virtual screening shows promising
shikimate kinase inhibition activity. The activity data of the synthesized
compounds suggests the importance of pyrazole moiety and amide
bond linking the pyrazole with the aryl group. The methyl substitutions
at R1-position is favorable for the activity and with small or large alkyl
substitutions at R-position also, it is clear that substitution at the pyr-
azole nitrogen with bulky groups decreases the activity. This confirms
the proposed methods have identified new scaffolds as inhibitors of
shikimate kinase. However, the IC50 values obtained for the new scaf-
folds were not as comparable with that of the lead molecule used for the
similarity search. This clearly suggests that further modification in the
molecule is required to improve the biological activity.
Compound 14
4. Conclusions
Compound 19–34
The aim of this study is to identify novel scaffold as inhibitors of
shikimate kinase on the basis of computational approach that can
predict putative inhibitors from a database of chemical compounds.
Utilizing ligand- and structure- based approaches similarity search and
molecular docking we selected ten compounds as inhibitors of
3.4. Biological testing
The synthesized compounds were tested for their potential for in-
hibiting the shikimate kinase enzyme using the method described
8

M.B. Rahul Reddy, et al.
BioorganicChemistry102(2020)104083
Fig. 5. List of selected compounds that can be a promising candidate for the inhibition of shikimate kinase.
Table 1
shikimate kinase. The compounds are selected on the basis of synthetic
feasibility and can be further explored along with their derivatives.
These new scaffolds identified confirm that the ligand- and structure-
based virtual screening based combined approach can possibly identify
promising candidates as inhibitors of shikimate kinase for the treatment
of tuberculosis.
Derivative compounds which are synthesized and shikimate kinase enzyme
inhibition activity of the synthesized compounds.
Compound No.
R
R1
Activity (IC50 in μg/mL)
14
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
–H
–CH3
–CH3
–NO2
–F
25
–H
25
–H
100
–H
50
Declaration of Competing Interest
–H
–CF3
–CH3
–NO2
–F
50
–C6H5
25
–C6H5
50
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
–C6H5
50
–C6H5
–CF3
–CH3
–NO2
–F
50
−2,4-diNO2C6H3
−2,4-diNO2C6H3
−2,4-diNO2C6H3
−2,4–diNO2C6H3
–CH3
50
greater than100
100
50
25
50
25
50
–CF3
–CH3
–NO2
–F
Acknowledgement
–CH3
–CH3
We are thankful to the Research council of SRMIST and The Dean,
SRM College of Pharmacy for providing support to carry out the work.
–CH3
–CF3
9

M.B. Rahul Reddy, et al.
BioorganicChemistry102(2020)104083
Appendix A. Supplementary material
M.T. Hamann, J. DeRuiter, D.C. Goodwin, A.I. Calderon, Slow-binding inhibition of
mycobacterium tuberculosis shikimate kinase by manzamine alkaloids,
Biochemistry 57 (2018) 4923–4933, https://doi.org/10.1021/acs.biochem.
8b00231.
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bioorg.2020.104083.
[17] P. Masoko, I.H. Mabusa, R.L. Howard, Isolation of alpha-linolenic acid from
Sutherlandia frutescens and its inhibition of Mycobacterium tuberculosis' shikimate
kinase enzyme, BMC Complement Altern. Med. 16 (2016) 366, https://doi.org/10.
1186/s12906-016-1344-1.
References
[18] R. Mehra, V.S. Rajput, M. Gupta, R. Chib, A. Kumar, P. Wazir, I.A. Khan,
A. Nargotra, Benzothiazole derivative as a novel mycobacterium tuberculosis shi-
kimate kinase inhibitor: identification and elucidation of its allosteric mode of in-
hibition, J. Chem. Inf. Model 56 (2016) 930–940, https://doi.org/10.1021/acs.
jcim.6b00056.
[1] World Health Organization (WHO). Global Tuberculosis report (2019), https://
www.who.int/tb/publications/global_report/en.
[2] S.S. Nilewar, M.K. Kathiravan, Mycothiol: A promising antitubercular target,
Bioorg. Chem. 52 (2014) 62–68, https://doi.org/10.1016/j.bioorg.2013.11.004.
[3] T.G. Benedek, The history of gold therapy for tuberculosis, J. Hist. Med. Allied Sci.
59 (2004) 50–89, https://doi.org/10.1093/jhmas/jrg042.
[19] V. Prado, E. Lence, M. Maneiro, J.C. Vazquez-Ucha, A. Beceiro, P. Thompson,
A.R. Hawkins, C. Gonzalez-Bello, Targeting the motion of shikimate kinase: de-
velopment of competitive inhibitors that stabilize an inactive open conformation of
the enzyme, J. Med. Chem. 59 (2016) 5471–5487, https://doi.org/10.1021/acs.
jmedchem.6b00483.
[4] Working Group on New TB Drugs pipeline, https://www.newtbdrugs.org/pipline.
php.
[5] K. Thomas. FDA Approves New Tuberculosis Drug. New York: The New York Times,
2012, https://www.nytimes.com/2013/01/01/business/fda-approves-new-tu-
berculosis-drug.html.
[20] S.K. Krishnasamy, V. Namasivayam, S. Mathew, R.S. Eakambaram, I.A. Ibrahim,
A. Natarajan, P. Senthilkumar, Design, synthesis, and characterization of some
hybridized pyrazolone pharmacophore analogs against Mycobacterium tubercu-
losis, Arch. Pharm. 349 (2016) 383–397, https://doi.org/10.1002/ardp.
201600019.
[6] A.C. Haagsma, R. Abdillahi-Ibrahim, M.J. Wagner, K. Krab, K. Vergauwen,
J. Guillemont, K. Andries, H. Lill, A. Koul, D. Bald, Selectivity of TMC207 towards
mycobacterial ATP synthase compared with that towards the eukaryotic homo-
logue, Antimicrob. Agents Chemother. 53 (2009) 1290–1292, https://doi.org/10.
1128/AAC.01393-08.
[21] M. Rarey, J.S. Dixon, Feature trees: A new molecular similarity measure based on
tree matching, J. Comput. Aided Mol. Des. 12 (1998) 471–490 https://link.
springer.com/article/10.1023/A:100806890462.
[7] A. Matteelli, A.C. Carvalho, K.E. Dooley, A. Kritski, TMC207: the first compound of
a new class of potent anti-tuberculosis drugs, Future Microbiol. 5 (2010) 849–858,
https://doi.org/10.2217/fmb.10.50.
[22] M. Rarey, M. Stahl, Similarity searching in large combinatorial chemistry spaces, J.
Comput. Aided Mol. Des. 15 (2001) 497–520 https://link.springer.com/content/
pdf/10.1023/A:1011144622059.
[8] J.H. Pereira, I.B. Vasconcelos, J.S. Oliveira, R.A. Caceres, W.F. de Azevedo,
L.A. Basso, L.S. Santos, Shikimate kinase: a potential target for development of
novel antitubercular agents, Curr. Drug Targets 8 (2007) 459–468, https://doi.org/
10.2174/138945007780059013.
[23] J.L. Durant, B.A. Leland, D.R. Henry, J.G. Nourse, Reoptimization of MDL keys for
use in drug discovery, J. Chem. Inf. Comput. Sci. 42 (2002) 1273–1280, https://doi.
org/10.1021/ci010132r.
[24] Molecular Operating Environment (MOE), 2018.01 (2018), Chemical Computing
Group ULC, 1010 Sherbooke St. West, Suite #910, Montreal, QC, Canada, H3A 2R7,
https://www.chemcomp.com/Products.
[9] T. Parish, N.G. Stoker, The common aromatic amino acid biosynthesis pathway is
essential in Mycobaterium tuberculosis, Microbiology 148 (2002) 3069–3077,
https://doi.org/10.1099/00221287-148-10-3069.
[25] J. Simithy, N. Reeve, J.V. Hobrath, R.C. Reynolds, A.I. Calderón, Identification of
shikimate kinase inhibitors among anti-Mycobacterium tuberculosis compounds by
LC-MS, Tuberculosis 94 (2014) 152–158, https://doi.org/10.1016/j.tube.2013.12.
004.
[10] R. Bentley, The shikimate pathway a metabolic tree with many branches, Crit. Rev.
Biochem. Mol. Biol. 25 (1990) 307–384, https://doi.org/10.3109/
10409239009090615.
[11] M.V.B. Dias, L.M. Faím, I.B. Vasconcelos, J.S. de Oliveira, L.A. Basso, D.S. Santos,
W.F de Azevedo, Effects of the magnesium and chloride ions and shikimate on the
structure of shikimate kinase from Mycobacterium tuberculosis, Acta Crystallogr.,
Sect. F: Struct. Biol. Cryst. Commun. 63 (2007) 1–6, https://doi.org/10.1107/
S1744309106046823.
[26] G. Timo, R. Reis, A. Melo, T. Costa, P. Magalhães, M. Homem de Mello, Predictive
power of in silico approach to evaluate chemicals against M. tuberculosis: A sys-
tematic review, Pharmaceuticals 12 (3) (2019) 135.
[27] H.M. Berman, J. Westbrook, Z. Feng, G. Gilliland, T.N. Bhat, H. Weissig,
I.N. Shindyalov, P.E. Bourne, The protein data bank, Nucleic Acids Res. 28 (2000)
235–242, https://doi.org/10.1093/nar/28.1.235.
[12] J.D. Coracini, W.F. de Azevedo, Shikimate kinase, a protein target for drug design,
Curr. Med. Chem. 21 (2014) 592–604, https://doi.org/10.2174/
09298673113206660299.
[28] J.S. Oliveira, C.A. Pinto, L.A. Basso, D.S. Santos, Cloning and overexpression in
soluble form of functional shikimate kinase and 5-enolpyruvylshikimate 3- phos-
phate synthase enzymes from Mycobacterium tuberculosis, Protein Expr. Purif. 22
(2001) 430–435, https://doi.org/10.1006/prep.2001.1457.
[13] S. Gordon, J. Simithy, D.C. Goodwin, A.I. Calder_on, Selective Mycobacterium tu-
berculosis Shikimate kinase inhibitors as potential antibacterials, Perspect. Med.
Chem. 7 (2015) 9–20, https://doi.org/10.4137/PMC.S13212.
[29] L.A. Rosado, I.B. Vasconcelos, M.S. Palma, V. Frappier, R.J. Najmanovich, D.S.
Santos, L.A. Basso, The mode of action of recombinant Mycobacterium tuberculosis
Shikimate kinase: kinetics and thermodynamics analyses, (2013), http://dx.doi.
org/10.1371/journal.pone.0061918.
[14] M.D. Hartmann, G.P. Bourenkov, A. Oberschall, N. Strizhov, H.D. Bartunik,
Mechanism of phosphoryl transfer catalyzed by shikimate kinase from
Mycobacterium tuberculosis, J. Mol. Biol. 364 (2006) 411–423, https://doi.org/10.
1016/j.jmb.2006.09.001.
[30] Y. Gu, L. Reshetnikova, Y. Li, Y. Wu, H. Yan, S. Singh, X. Ji, Crystal structure of
shikimate kinase from Mycobacterium tuberculosis reveals the dynamic role of the
LID domain in catalysis, J. Mol. Biol. (2002) 779–789, https://doi.org/10.1016/
S0022-2836(02)00339-X.
[15] V.S. Rajput, R. Mehra, S. Kumar, A. Nargotra, P.P. Singh, I.A. Khan, Screening of
antitubercular compound library identifies novel shikimate kinase inhibitors of
Mycobacterium tuberculosis, Appl. Microbiol. Biotechnol. 100 (2016) 5415–5426,
https://doi.org/10.1007/s00253-015-7268-8.
[16] J. Simithy, N.R. Fuanta, M. Alturki, J.V. Hobrath, A.E. Wahba, I. Pina, J. Rath,
10