Homology modelling, molecular docking, and molecular dynamics simulations reveal the inhibition of Leishmania donovani dihydrofolate reductase-thymidylate synthase enzyme by Withaferin-A
Vadloori et al. BMC Res Notes
Homology modelling, molecular docking, and molecular dynamics simulations reveal the inhibition of Leishmania donovani dihydrofolate reductase-thymidylate synthase enzyme by Withaferin-A
Bharadwaja Vadloori 0
A. K. Sharath 1
N. Prakash Prabhu 1
Radheshyam Maurya 0
0 Department of Animal Biology, School of Life Sciences, University of Hyderabad , Prof. C.R. Rao Road, Gachibowli, Hyderabad 500046 , India
1 Department of Biotechnology and Bioinformatics, University of Hyderabad , Hyderabad , India
Objective: Present in silico study was carried out to explore the mode of inhibition of Leishmania donovani dihydrofolate reductase-thymidylate synthase (Ld DHFR-TS) enzyme by Withaferin-A, a withanolide isolated from Withania somnifera. Withaferin-A (WA) is known for its profound multifaceted properties, but its antileishmanial activity is not well understood. The parasite's DHFR-TS enzyme is diverse from its mammalian host and could be a potential drug target in parasites. Results: A 3D model of Ld DHFR-TS enzyme was built and verified using Ramachandran plot and SAVES tools. The protein was docked with WA-the ligand, methotrexate (MTX)-competitive inhibitor of DHFR, and dihydrofolic acid (DHFA)-substrate for DHFR-TS. Molecular docking studies reveal that WA competes for active sites of both Hu DHFR and TS enzymes whereas it binds to a site other than active site in Ld DHFR-TS. Moreover, Lys 173 residue of DHFR-TS forms a H-bond with WA and has higher binding affinity to Ld DHFR-TS than Hu DHFR and Hu TS. The MD simulations confirmed the H-bonding interactions were stable. The binding energies of WA with Ld DHFR-TS were calculated using MM-PBSA. Homology modelling, molecular docking and MD simulations of Ld DHFR-TS revealed that WA could be a potential anti-leishmanial drug.
Leishmania donovani; DHFR-TS; Withania somnifera; Ashwagandha; Molecular docking; Withaferin-A; Methotrexate; Dihydrofolicacid; Antileishmanial drug
Withaferin-A (WA) is among the most effective
withanolide isolated from W. somnifera and has various effects
like anti-bacterial, anti-inflammatory, anti-proliferative
and potent anti-cancer properties [
]. Recently we
demonstrated in vitro, that withanolides show potent
anti-leishmanial activity  and a drastic reduction in
parasite load in vivo [
Availability of complete genome sequence of
Leishmania opens new windows to identify a potential drug
]. Many enzymes of Leishmania are extensively
explored as drug targets as they are diverse from
mammalian hosts [
]. Trypanosomatids including
Leishmania are pteridine auxotrophs and require an exogenous
source of folate/biopterin [
]. Folate and biopterin
are served as cofactors only in their fully reduced forms,
H4-folate and H4-biopterin, respectively (Fig. 1a). In
Leishmania DHFR along with TS forms DHFR-TS
complex and occurs as a bifunctional enzyme [
However, as de novo biopterin synthetic pathway is absent,
DHFR-TS shows no activity with biopterin [
Parasite obtains folates from the host and uses its
DHFRTS and PTR1 enzymes to reduce folates to active H4
Hence, folate biosynthesis enzymes can be potential
drug targets and molecules which inhibit any enzyme of
these pathways can be a safe antileishmanial drug. Our in
silico study shows that WA inhibits multiple enzymes in
folate biosynthesis pathway of Leishmania parasites.
Amino acid sequences of Ld DHFR-TS, (accession no.
CBZ31672.1, Homo sapiens or Human DHFR (Hu DHFR)
(AAH71996.1) and Homo sapiens or Human TS (Hu
TS) (NP_001062.1) were obtained from NCBI (http://
www.ncbi.nlm.nih.gov). The similarity in sequences
between host and parasite enzymes was identified using
Clustal omega (https://www.ebi.ac.uk/Tools/msa/clust
alo/). Template for structural modeling was identified
using PDB-BLAST. Protein model was developed using
SWISS-Model (https://swissmodel.expasy.org/) [
and verified with Ramachandran plot, PROCHECK
analysis, global model quality estimation (GMQE) score
and qualitative model energy analysis (QMEAN) values
The structures of WA, MTX and DHFA (PubChem CID
265237, 126941, 98792, respectively) were obtained from
(Additional file 1: Fig. S1). Open Babel (http://openbabel.
org/wiki/Main_Page) was used to obtain. pdbqt files.
Molecular docking studies were carried out in Auto Dock
]. Initially, blind docking, was performed,
followed by docking within restricted search space around
the probable binding sites. Docking conformations were
selected based on binding affinity. Pymol (https://www.
pymol.org/) was used for visualization and graphical
Drug-likeness of WA [
] was calculated using
molsoft server (http://molsoft.com/mprop/). A drug-likeness
plot and score were obtained. Swiss target was used to
predict drug target class for Withaferin A. The server,
using a combination of 2D and 3D similarity measures,
compares the query molecule to a library of 280,000
compounds active on more than 2000 targets of five
different organisms .
Molecular dynamic simulation
MD simulation of Ld DHFR-TS, Hu DHFR and Hu TS,
and their WA complexes were performed in Gromacs 5.0.
]. The topological
parameter of the ligand was obtained from ATB server (https
]. Initially, protein or its complex
was kept in a cubic box filled with water using SPC/E
water models. The system was energy minimized using
GROMOS54a7 force field [
] and equilibrated at 300 K
using V-rescale for 200 ps as NVT ensemble followed by
equilibration at 1 atm pressure using Parrinello–Rahman
algorithm as NPT ensemble for 200 ps. The equilibrated
conformation was further extended for production
simulation for 25 ns. LINCS algorithm was applied for bond
constraints with distance cut-off using Verlet during
simulation. Root mean square deviations of atomic
coordinates during the simulation from their respective
initial coordinates were calculated using the gmx_rms tool
in Gromacs and binding energies were calculated using
Sequence alignment and homology modeling
The sequence similarity between Hu DHFR and Ld
DHFR-TS was found to be 25.13%, and between Hu
TS and Ld DHFR-TS, it was 54.63% suggesting that Ld
DHFR-TS could be a valid drug target (Additional file 1:
Figs. S2, S3). The amino acid sequence of Ld DHFR-TS
was blasted against PDB-BLAST database for identifying
an appropriate template for homology modeling. T.cruzi
DHFR-TS showed 67.32% identity with the target
protein and was selected as a template (Additional file 1: Fig.
S4). Quality of the model generated by Swiss-model was
verified using different tools (Fig. 1b) (Additional file 1:
Table S1). The selected model showed 0.2% of residues
in disallowed regions of Ramachandran plot (Additional
file 1: Fig. S5, Table S2) with GMQE score of 0.82 and
QMEAN score of − 2.25 (Additional file 1: Fig. S6).
The generated model is a homo-dimer protein of α+ β
class. The protein consists of 4β-sheets, 3βαβ units,
5β-hairpins, 19β-strands, 21α-helices (Additional file 1:
Fig. S7). Similar numbers of secondary structural
elements were found in T. cruzi DHFR-TS and RMSD
between the template and generated model was
calculated to be 0.625 Å.
Drug‑likeness of Withaferin A
A compound to be considered as a drug should
have ≤ 5 H-bond donors (HBD), ≤ 10 H-bond acceptors
(HBA), molecular weight (MW) ≤ 500 Daltons, octanol–
water partition coefficient (Log P) value between − 0.4
to + 5.6, and polar surface area (PSA) ≤ 140 Ǻ2 [
has 2HBDs, 6HBAs, MW of 470.27, Log P of 3.21, and
PSA of 75.66 A2. The drug-likeness model score was 0.36
(Additional file 1: Table S3). Further, the frequency of
drug target class as predicted by Swiss target prediction
for WA is enzymes (40%) and kinases (33%).
Molecular docking studies
To know the active site of Ld DHFR-TS, it was first
docked with its substrate DHFA and found that it has
two active sites, one in DHFR and other in TS domain.
TS active site is located 40 Å away from DHFR active
]. Asp 52, Arg 97 and Thr 180 of DHFR
domain form H-bonds with DHFA and binding energy is
− 29.3 kJ/mol. Arg 283, His 401, Gln 421, and Asn 433
of TS domain form H-bonds with DHFA and binding
energy is − 31.8 kJ/mol.
MTX is a known competitive inhibitor of DHFR, hence
Ld DHFR-TS was also docked with MTX. The results
show that MTX binds at active sites (Fig. 1c). Ser 86 of
DHFR domain forms H-bond with MTX and binding
energy is − 33.1 kJ/mol. Arg 283, Glu 292, His 401, Gln
421 and Asn 433 of TS domain form H-bonds with MTX
and binding energy is − 31.8 kJ/mol. The Binding site
for MTX was compared with a 3D crystal structure of
bifunctional Tc DHFR-TS in complex with MTX (3CL9)
by superimposing on Ld DHFR-TS docked with MTX
and RMSD of the ligand was found to be 0.625 Å.
Likewise, crystal structure of mouse TS in ternary complex
and cofactor product, dihydrofolate (4EZ8), crystal
structure of Hu TS, ternary complex with dUMP and
tomudex (1i00) and Hu TS in complex with dUMP and MTX
(5 × 66) were also used for superimposing and
confirming the respective positions of ligands. RMSD values were
0.768, 0.806 and 0.669 Å respectively. Further, Ld
DHFRTS was docked with WA and Lys 173 forms an H-bond
with WA. The binding energy of WA is − 42.7 kJ/mol
and it binds in between both the active sites. It blocks the
electrostatic channel of the enzyme (Fig. 1c).
Crystal structure of Hu DHFR (4m6k) was docked
with WA and was superimposed with Hu DHFR ternary
crystal complex of MTX and NADPH (1u72) and
crystal structure of Hu DHFR complex of NADP+ and folate
(4m6k). The results showed all three ligands viz. WA,
DHFA, and MTX are binding in the same pocket. The
ligand WA also competes for the active site and might be
acting as a competitive inhibitor. The binding energy of
WA is − 41.4 kJ/mol (Fig. 1d).
Crystal structure of Hu TS (1hzw) was docked with
WA and later superimposed with Hu TS complex of
dUMP and MTX (5x66). We observed that WA is
binding at the same site like MTX. The residues Phe 80, His
196, Leu 221 and Asn 226 were forming H-bonds with
WA and binding energy of WA was − 39.8 kJ/mol. The
ligand WA was again competing for the active site and
might be acting as a competitive inhibitor (Fig. 1e). Lys
173 forms an H-bond with WA. No H-bonding with WA
was observed in Hu DHFR and Phe 80, His 196, Leu 221
and Asn 226 form H-bonds with WA in Hu TS. Although,
WA is not binding in the active site of Ld DHFR-TS, it
binds to human enzymes due to differences in the
The docking results of Hu DHFR and TS with WA
suggest that WA competes for substrate binding sites of both
human enzymes and act as competitive inhibitor. In case
of Ld DHFR-TS, WA act as an uncompetitive inhibitor.
The binding energy of Ld DHFR-TS with WA is higher
than Hu DHFR and TS. Moreover, WA could be a better
drug than MTX because of its high binding energy.
Molecular dynamic simulations of enzyme‑inhibitor complexes
To characterize the stabilizing interactions and to
evaluate binding energies of WA with Ld DHFR-TS, Hu DHFR
and Hu TS, MD simulation of proteins and protein-WA
complexes were carried out. The analysis of root mean
square deviations (RMSD) showed all proteins attained
almost stable conformations (Fig. 2a–c) with
comparable RMSD values. Addition of WA did not show much
change in RMSD of Hu DHFR whereas RMSD of Ld
DHFR-TS slightly increased. RMSD of ligand alone was
around 0.15 nm in all proteins suggesting that bound
conformation was stable. Further, root mean square
fluctuations (RMSF) of individual residues were calculated
by considering their Cα atoms as a reference (Fig. 2d–
f ). RMSF of β5-loop in DHFR domain and β1′ and β4′
− 130.454 ± 11.349
− 49.324 ± 14.361
122.969 ± 30.393
− 15.405 ± 2.536
− 72.214 ± 18.570
Types of energy (kJ/mol)
Van der Waal energy
Polar solvation energy
Non-polar solvation energy
loops in TS domain were found to increase slightly in
the ligand-bound state of Ld DHFR-TS. The RMSF of β4
and β6 loops of DHFR domain reduced. In WA bound
Hu DHFR, the fluctuations around β2, β3, and β6 loops
reduced. In case of WA bound Hu TS protein, RMSF of
β1 loop reduced whereas β3 increased. In all three
proteins, changes in fluctuations were observed largely at
sites away from ligand binding sites. Moreover,
during binding of WA with Ld DHFR-TS, it was observed
that WA formed H-bonding interactions with a
backbone of F483 and side chains of Arg275, Asn199, and
Asn231. Similarly, H-bonding interactions were
identified between WA and backbone of Gly7 and side chain of
Gln48 in Hu DHFR. WA formed H-bonding interactions
with Arg163 and I1e 78 of Hu TS.
For further quantitative binding, energies of ligand
were calculated by MM-PBSA using the last 10 ns of
simulation data where RMSD of proteins were found to
be more stable (Table 1). The analysis indicates that
binding affinity of WA is more towards Ld DHFR-TS than Hu
DHFR or Hu TS.
Interestingly, Leishmania dhfrts− mutants are unable to
survive in mammalian host [
]. Deletion of PTR1 gene
is lethal in promastigotes, indicating an essential role
for unconjugated pteridines [
expression provides a potential ‘metabolic by-pass’ of
DHFRTS inhibition and allows a partial or complete reversal of
anti-pteridine inhibition in the promastigote stage of
]. PTR1 activity in L. major promastigotes
is lower than in L. donovani and L. mexicana. L. major
is more sensitive to MTX suggesting the role of PTR1 as
a metabolic-bypass in L. donovani and L. mexicana [
]. 3D structures of DHFR-TS and PTR1 of parasite and
Hu DHFR have provided a strong base to design new
inhibitors which are selective for parasite alone [
Recently, we reported that WA inhibits Ld PTR1
enzyme activity and molecular docking studies of WA
showed high binding affinity with PTR1. Enzyme assay
with purified PTR-1 revealed that WA inhibits enzyme
activity through uncompetitive mode [
]. The present
molecular docking study reveals that the binding energy
of WA with Ld DHFR-TS is higher than Hu DHFR, Hu
TS enzymes and WA inhibits Ld DHFR-TS same as the
PTR-1 enzyme. Thus it could be concluded that
binding affinity of WA with multiple enzymes (DHFR-TS and
PTR1) of folate biosynthesis pathway of parasites could
make WA an effective anti-leishmanial drug.
Due to the lack of purified DHFR-TS enzyme, the current
study could not include enzyme assay. However, enzyme
assayed from parasite lysate with WA has shown the
inhibition activity reported earlier [
Additional file 1: Figure S1. The structures of ligands: (A) Withaferin-A,
(B) Methotrexate, (C) DHFA drawn using Chemdraw ultra version 12.0
software. Figure S2. Sequence identity between Hu DHFR and Ld
DHFRTS. Asterisks indicate identical amino acids. Dots and colons indicate
conserved amino acid substitutions. Dashes indicate gaps. Figure S3.
Sequence identity between Hu TS and Ld DHFR-TS. Asterisks indicate
identical amino acids. Dots and colons indicate conserved amino acid
substitutions. Dashes indicate gaps. Figure S4. Sequence identity between
Ld DHFR-TS and T.cruzichain A. Asterisks indicate identical amino acids.
Dots and colons indicate conserved amino acid substitutions. Dashes
indicate gaps. Figure S5. Ramachandran Plot: (A) Modelled Ld DHFR-TS
and (B) reference T. cruzi DHFR-TS obtained using PROCHECK. Figure S6.
Local quality estimate of (A) modelled Ld DHFR-TS and (B) reference T.cruzi
DHFR-TS obtained from Swiss model. Figure S7. Secondary structures of
(A) modelled Ld DHFR-TS and (B) reference T.cruzi DHFR-TS obtained from
PDB sum. Table S1. Features of the generated Ld DHFR-TS model from
Swiss model. Table S2. Ramachandran plot Statistics from PROCHECK
results for modelled Ld DHFR-TS protein and reference T. cruzi DHFR-TS
protein. Table S3. Drug likeness properties of WA from molsoft.
2D: two dimensional; 3D: three dimensional; Å: Angstrom; ATB: automated
topology builder; atm: atmosphere; DHFA: dihydrofolic acid; GMQE: global
model quality estimation; HBA: hydrogen bond acceptors; HBD: hydrogen
bond donors; Hu DHFR: human dihydrofolate reductase; Hu TS: human
thymidylate synthase; K: Kelvin; Ld DHFR-TS: Leishmania donovani
dihydrofolate reductase-thymidylate synthase; LINCS: LINear constraint solver; Log P:
octanol–water partition coefficient; MDS: molecular dynamic simulations;
MM-PBSA: molecular mechanics-Poisson–Boltzmann surface area; MTX:
methotrexate; MW: molecular weight; nm: nano meters; ns: nano seconds;
NCBI: National Center for Biotechnology Information; NPT ensemble:
isothermal (constant temperature T)-isobaric (constant pressure P) ensemble; NVT
ensemble: number of particles (N), absolute temperature (T) and volume (V)
ensemble; PDB-BLAST: Protein Data Bank-Basic Local Alignment Search Tool;
ps: pico seconds; PSA: polar surface area; PTR: pteridine reductase; QMEAN:
qualitative model energy analysis; RMSD: root mean square deviation; RMSF:
root mean square fluctuations; SAVES: structure analysis and verification
server; SPC/E water models: extended simple point charge model; Tc DHFR-TS:
Trypanosoma cruzi dihydrofolate reductase-thymidylate synthase; V-rescale:
velocity rescale; WA: Withaferin A.
RM and BV conceived the idea and designed the experiments. BV and AKS
performed the in silico experiments. BV, AKS, PP, and RM analyzed and
interpreted data. PP and RM corrected and edited the manuscript. All authors read
and approved the final manuscript.
We sincerely acknowledged the bioinformatics facilities DBT-BINC, DST-PURSE,
DBT-CREB and BBL fellowship to Mr. Bharadwaja Vadloori from University of
The authors declare that they have no competing interests.
Availability of data and materials
The PDB file of the Ld DHFR-TS enzyme model (PMDB ID: PM0081119)
generated by homology modelling has been deposited in the protein model
database repository. (https://bioinformatics.cineca.it/PMDB/user/graph_model
Consent for publication
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Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
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