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Archana Moon*et al. /International Journal of Pharmacy & Technology
IJPT| April-2017| Vol. 9 | Issue No.1 | 28816-28829 Page 28816
ISSN: 0975-766X
CODEN: IJPTFI
Available Online through Research Article
www.ijptonline.com IN SILICO STUDIES OF INHIBITORS OF DIHYDROFOLATE REDUCTASE AND DIHYDROPTEROATE
SYNTHASE OF E.COLI
Archana Moon1, Deeba Khan
2, Pranjali Gajbhiye
3 & Monali Jariya
4
1,2,3&4 University Department of Biochemistry, Rashtrasant Tukadoji Maharaj Nagpur University, Nagpur -440033.
Email: [email protected]
Received on: 10-02-2017 Accepted on: 22-03-2017
Abstract:
In the past two decades, antibiotic resistance has become an increasingly severe health problem. Bacterial infections
caused by resistant strains are problematic in numerous hospitals around the world, especially in patients compromised
by age, illness, and treated with immune-suppressant drugs. Antibiotics have a distinct mechanism of action capable of
modifying microbial metabolism and physiological processes such as translation, DNA replication and cell wall
biosynthesis. In this context, it is essential to increase the understanding of resistance mechanisms in order to develop
drugs with potential activity against these pathogens. Escherichia coli is the most common causative agent of urinary
tract infection (UTI), and diagnosing this infection usually relies on bacteriologic methods. Urinary tract infection (UTI)
is the second most common bacterial infection after respiratory tract infection. Approximately 75-95% of UTIs are
caused by Escherichia coli. Sulfonamides are competitive inhibitors of dihydropteroate synthase, the bacterial enzyme
responsible for the incorporation of para-aminobenzoic acid (PABA) into dihydropteroic acid, the immediate precursor
of folic acid.
Trimethoprim exerts a synergistic effect with sulfonamides. It is a potent and selective competitive inhibitor of
microbial dihydrofolate reductase, the enzyme that reduces dihydrofolate to tetrahydrofolate. Docking studies are
molecular modelling studies aimed at finding a proper fit between ligand and its binding site. The present paper deals
with studies to corrobarate the microbial and molecular studies to treat MDR resistant bacterial infections. Autodock
Suite, version 1.5 6rC2 for Docking has been utilized in this study.
Keywords: Dihydrofolate reductase(DHFR), Dihydropteroate synthase (DHPS), Docking, E.coli, Multidrug
resistant(MDR).
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Introduction:
UTI is a serious health problem affecting millions of people each year. Infections of the urinary tract are the second most
common type of infection. These are one of the most common bacterial infections affecting humans throughout their
lifespan. These infections are more common in females than in males (1). It is caused by Gram-negative bacteria such as
Escherichia coli, Klebsiella species, Enterobacter species, Proteus species and Gram-positive bacteria like Enterococcus
species, and Staphylococcus saprophyticus. Studies from various parts of India have shown occurrence of high rates of
antimicrobial resistance among E coli. The resistance rates of uropathogenic E. coli to various antibiotics such as beta-
lactams (57.4%), co-trimoxazole (48.5%), quinolones (74.5%), gentamicin (58.2%), amikacin (33.4%), cefuroxime
(56%), nalidixic acid (77.7%) have been reported (2). UTI due to multi drug resistant (MDR) E. coli increases the cost of
treatment, morbidity and mortality especially in developing countries like India (2). The infection caused by MDR
bacteria leads to limited treatment options (3). Antibiotic resistance is a worldwide problem threatening the ability to
treat infections (4). Despite advancements of higher generations of antibiotics, these antibacterial agents have not been
up to the mark in eradicating these infectious diseases. One of the possible reasons responsible for this decline in
therapeutic control of the disease is antibiotic resistance. The MDR bacteria due to continued mismanaged selective
pressure have been credited for being responsible for genetic response to antibacterial therapy (5).
Resistance in Gram-negative bacteria has been increasing, particularly over the last 6 years. Many of the isolates
producing these enzymes are also resistant to trimethoprim, quinolones and aminoglycosides. Novel combinations of
antibiotics are being used in the community and broad-spectrum agents such as carbapenems are being used increasingly
as empirical treatment for severe infections (6). Microorganisms use various mechanisms to develop drug resistance,
such as recombination of foreign DNA in bacterial chromosome, horizontal gene transfer and alteration in genetic
material (7). The folate metabolic pathway leads to synthesis of required precursors for cellular function and contains a
critical node, dihydrofolate reductase (DHFR), which is shared between prokaryotes and eukaryotes. The DHFR enzyme
is currently targeted by methotrexate in anti-cancer therapies, by trimethoprim for antibacterial uses, and by
pyrimethamine for anti-protozoal applications. An additional anti-folate target is dihyropteroate synthase (DHPS), which
is unique to prokaryotes as they cannot acquire folate through dietary means. It has been demonstrated as a primary
target for the longest standing antibiotic class, the sulfonamides, which act synergistically with DHFR inhibitors.
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Investigations have revealed most DHPS enzymes possess the ability to utilize sulfa drugs metabolically, producing
alternate products that presumably inhibit downstream enzymes requiring the produced dihydropteroate. These inhibitors
are also likely to interact with the enzymatic neighbors in the folate pathway that bind products of the DHFR or DHPS
enzymes and/or substrates of similar substructure (8) Fig (1). Commonly used sulfa drug is co-trimoxazole, an
association of two antifolate agents, sulfamethoxazole and trimethoprim (9). Dihydrofolate reductase (DHFR) is a
ubiquitous enzyme present in almost all eukaryotic and prokaryotic cells (11). Inhibition of DHFR results in a depletion
of the reduced folate pool, inhibition of RNA and DNA synthesis, and cell death. For this reason, DHFR has been a
critically important therapeutic drug target. DHFR inhibitors targeting the FH2 binding site have been used in the
treatment of cancer, autoimmune diseases and bacterial and fungal infections (10). Most microorganisms must synthesize
folate denovo since they lack the active transport system of higher vertebrate cells that allows these organisms to use
dietary folates. DHPS (Gene folP) is the target of sulphonamides, which are substrate analogues that compete with para-
aminobenzoic acid. Molecular docking has become an increasingly important tool for drug discovery (12). Detailed
analysis was performed for a better understanding of the molecular interactions between the ligands and target DHFR &
DHPS proteins of E. coli based on their efficiency to bind the active sites on the receptor utilizing Autodock (version 1.5
6rC2). The docking exhibits different binding poses out of which the one with minimum binding energy was selected.
The favourable conformation was analysed with hydrogen bonding with the active site residues of DHFR & DHPS (13).
Fig(1): Folate Pathway In Bacteria (20).
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Materials & Methods:
In silico studies
Molecular docking is an important tool to study the interaction of ligands with active site residues of the receptor [14,
15]. The docking involves the use of sampling algorithm and a scoring function to evaluate the proper orientation and
pose of ligand molecule in relation to the binding energy. The correct identification of this binding pose of one or more
related ligands is important in establishing a structure-activity relationship in lead optimization. The second use of
scoring functions is to rank different ligands to predict their relative experimental activity [15-17].
In silico studies were performed using Autodock 4 suite (version 1.5 6rC2). The ligands viz, Chlorogenic acid, Ellagic
acid, Gallic Acid, Hippuric acid, Quercetin and Standard Antibiotics viz, Clavulanic acid, Cephalosporin, Cephalosporin
C, Penicillin, Sulfamethoxazole and Trimethoprim were docked with DHFR & DHPS enzymes of E.coli. The structure
of DHFR of E.coli was downloaded from PDB (protein data base) with PDB Id 2ANO (X ray diffraction structure,
2.6A0) & PDB Id 1AJ0 for DHPS of E.coli ( X ray diffraction structure, 2.0A
0). Refer Fig (2). The PubSum database,
utilizes 4 characters, as inputs, to get the ligands with their Ligplots. Ligplots indicate the interacting sites of the protein
of interest i.e DHFR & DHPS Fig(3,4). Next a Ramachandran Plot Fig(5) to verify the protein structure was plotted. The
structure of the ligands were downloaded from Pubchem (chemical structure data base) online portal and drawn in
Marvin Sketch version 5.8.1. Fig(6,7). Docking was performed for DHFR & DHPS of E.coli with ligands (Chlorogenic
acid, Ellagic acid, Gallic Acid, Hippuric acid and Quercetin and Standard Antibiotics (Clavulanic acid, Cephalosporin
Cephalosporin C, Penicillin, Sulfamethoxazole and Trimethoprim). The docking results were analyzed on the basis of
their binding energy and their interactions (13).
Fig(2): PDB Structure of DHFR and DHPS Protein.
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Fig(3): Ligplot of DHFR E.coli Ligand: A)Ligand NDP- (Ala7, Ile14, Arg44, Thr46, Ser63, Ser64, Gly97, Arg98,
Gln102) &B) Ligand 817 – Asp27 & Thr113 interaction are shown by green dashed line.
Fig(4): Ligplot of DHPS E.coli Ligand: A)Ligand SO4 284 –Arg255, Asn22, Arg63, His257 B) Ligand PH2 559-
Thr62, Lys221, Asn115, Asp96 & C) San561 – SER219 interaction are shown by green dashed line.
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Fig (5): The Ramachandran plot shows the phi-psi torsion angles for all residues in the structure. Glycine residues
are separately identified by triangles as these are not restricted to the regions of the plot appropriate to the other
sidechain types. The colouring/shading on the plot represents the different regions: the darkest areas (here shown
in red) correspond to the "core" regions representing the most favourable combinations of phi-psi values.
Fig(6): INHIBITORS - (A) Chlorogenic acid,(B) Ellagic acid, (C) Gallic acid, (D) Hippuric acid and (E)
Quercetin.
Fig(7): ANTIBIOTICS AS LIGANDS- (A)Clavulanic acid, (B) Cephalosporin,(C) CephalosporinC, (D) Penicillin,
(E) Sulfamethoxazole and (F) Trimethoprim
Preparation of Proteins and Ligands:
The Ligands that exhibited prominent antibacterial activity towards isolated multidrug-resistant bacteria were selected
for molecular docking analysis. The structure of DHFR & DHPS was opened in Biovia Discovery Studio 2016 version
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16.1.0.15350. The structure of protein was cleared (i.e. the extra groups which includes water molecules, ligand groups
were removed) by deleting the heteroatoms present in the protein (18). Only the protein and active site for docking is
required. Hence was saved in the PDB format. The structure of ligands were downloaded from Pubchem and drawn in
Marvin Sketch view version 5.8.1 and cleaned in 2D and 3D. This clears the 2 dimensional and 3 dimensional structure
of the ligand. For docking, the protein structure was obtained in PDB format and ligands in tripos-Mol format or PDB
format (18).
Grid formation by Autodock:
Grid points generate the coordinates or interaction points where the ligand is docked. The grid box was generated at
60x60x60 A0
to cover all the active site residues, and allowed the flexible rotation of ligands. The GA (genetic
algorithm) and number of generation were set to 10 and 27000 for DHFR 60 and 27000 for DHPS respectively. The
Lamarckian genetic algorithm was followed for ligand confirmation. All the above parameters decide the different
confirmation of ligand in which the ligand will be docked. Other parameters for example, free energy (after docking is
complete we get the value of free energy), rotatable bonds (number of rotatable bonds varies according to the ligand
structure), number of torsions (18) etc were used as default (18).
Result:
Docking studies revealed the interaction of the protein with the ligand, binding energy, type of interaction and amino
acids involved in interactions. Binding energy should be negative. More negative the binding energy, better the binding
affinity of ligand and protein (18). Table 1 & 2 give the binding energy of ligands with DHFR & DHPS respectively.
Table No.1: Binding Energy of Legends with DHFR Protein of E.COLI.
LIGANDS Binding Energy of DHFR E.coli
INHIBITORS
Chlorogenic acid -8.70
Ellagic acid -7.90
Gallic acid -5.93
Hippuric acid -6.45
Quercetin -7.73
ANTIBIOTICS
Clavulanic acid -6.41
Cephalosporin -8.48
Cephalosporin C -9.10
Penicillin -9.11
Sulfamethoxazole -7.50
Trimethoprim -7.91
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Inhibitors (Chlorogenic acid, Ellagic acid, Gallic Acid, Hippuric acid and Quercetin) and Standard Antibiotics
(Clavulanic acid, Cephalosporin, Cephalosporin C, Penicillin, Sulfamethoxazole and Trimethoprim) were docked and the
results obtained provide a comparative insight into the potency of inhibitors and Standard Antibiotics through analysis of
their binding capacities. The binding energies of DHFR of E.coli with Penicillin, Cephalosporin C & Chlorogenic acid
are better than other ligands. From table no.1 it is clear that Standard Antibiotics like Penicillin and Cephalosporin C are
more effective than the inhibitors docked. Table No.2:
Table No.2: Binding Energy of Legends with DHPS Protein of E.COLI.
The binding energies of DHPS of E.coli with Clavulanic acid, Gallic acid, Hippuric acid and Trimethoprim are (-6.57,-
6.25,-5.91 and -5.58). From table no.2 it is clear that Standard Antibiotics like Clavulanic acid are more effective than
the inhibitor i.e Gallic acid and Hippuric acid for the DHPS protein.
Table No.3: Interaction of ligands with dhfr and dhps protein i.e. Hydrogen bond length and name and amino
acid involved in interaction.
LIGANDS DHFR E.coli DHPS E.coli
Hydrogen
Bond Length
In A°
Hydrogen
Bond Name
Interactin
g Sites
Hydrogen
Bond Length
In A°
Hydrogen
Bond Name
Interacting
Sites
Chlorogenic 2.202 Thr46 Ala7 1.969 Asn115 Asn115
LIGANDS Binding Energy of DHPS E.coli
INHIBITORS
Chlorogenic acid +4.98
Ellagic acid -2.31
Gallic acid -6.25
Hippuric acid -5.91
Quercetin -4.43
ANTIBIOTICS
Clavulanic acid -6.57
Cephalosporin +8.94
Cephalosporin C +0.42
Penicillin +6.13
Sulfamethoxazole -3.99
Trimethoprim -5.58
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acid 2.083
1.846
1.888
2.205
Thr46
Gly97
-
Ala7
1.824 - Arg255
Ellagic acid 2.125
1.79
2.173
2.233
-
-
-
Ser49
Thr46
Ala7
2.143 - -
Gallic acid 1.838
2.018
1.993
-
-
Ala7
Asp27 1.959 - -
Hippuric acid 2.148 Ala7 Thr113
Ala7
1.978 - -
Quercetin 1.725
1.963
Gly97
Thr123
Ser64
Gln102
Arg98
1.727
1.782
-
Arg255
Lys221
Clavulanic acid 1.978
2.212
-
Ala7
Ala7 2.035
1.655
2.11
1.83
Asn115
Lys221
Phe188
-
Lys221
Asp185
Cephalosporin 2.242
2.146
Gly97
Ala7
Ala7 1.738 Ser94 Thr62
Cephalosporin
C
2.1
2.135
Arg98
Asn18
Gly97
Arg98
- No -
Penicillin 2.171
1.907
Ala7
Gly97
Thr46
Gly97
1.693 Gly191 -
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Ala7
Sulfamethoxaz
ole
2.127
1.938
-
Ala7
Thr46
Ala7
1.919 Met223 -
Trimethoprim 1.878
1.835
-
-
Ala7 1.827 - -
Table 3 give the interaction of various ligands with DHFR & DHPS i.e. hydrogen bond length, hydrogen bond name and
amino acid involved in the interaction. As these ligands/ inhibitors have proven antibacterial, antimicrobial and
anticancer activity, these ligands can be further used as lead compounds in treatment of multidrug resistant urinary tract
infection caused by E.coli.
Inhibitors (EA, GA, HA & Q) & Standard Antibiotics (Cep C, Pen, SMX &TMP) show no interaction with DHPS. This
indicates that the ligands do not bind to the active site of the protein. Cep C shows no hydrogen bonding, which indicates
improper docking.
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Fig (8): Interacting Sites of DHFR with Inhibitors i.e. (A) Chlorogenic acid,(B) Ellagic acid, (C) Gallic acid, (D)
Hippuric acid and (E) Quercetin.
Fig(9): Interacting Sites of DHFR with Standard Antibiotics i.e. (A) Clavulanic acid, (B) Cephalosporin,(C)
CephalosporinC, (D) Penicillin, (E) Sulfamethoxazole and (F) Trimethoprim.
Fig(10): Interacting Sites of DHPS with Inhibitors and Standard Antibiotics: (A) Chlorogenic acid, (B) Quercetin,
(c) Clavulanic acid and (D) Cephalosporin.
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Discussion:
Urinary tract infections (UTIs) are among the most common infections in humans. The majority of cases are caused by a
limited number of bacterial genera; E. coli strains in particular are responsible for 80% of the UTI cases seen in
outpatient clinics. These strains are found in the normal flora of the intestinal tract, skin, and vagina.(19)
Insilico studies with DHFR & DHPS showed various hydrogen interactions with inhibitors viz., Chlorogenic acid,
Ellagic acid, Gallic Acid, Hippuric acid and Quercetin and Standard Antibiotics viz., Clavulanic acid, Cephalosporin,
Cephalosporin C, Penicillin, Sulfamethoxazole and Trimethoprim.
From the results, it is clear that the inhibitors CGA, EA, GA, HA and Q show negative/higher binding affinity. These
were analysed for their binding efficiency to the active site of DHFR of E.coli. As compared to inhibitors particularly
CGA, antibiotics show higher binding results, such as Penicillin & Cephalosporin C, thereby contributing towards higher
interactions with the DHFR of E.coli.
Our study reveals that inhibitors like Clavalunic acid and Gallic acid were analysed for their binding efficiency to the
active site DHPS of E.coli. Clavalunic acid and Gallic acid have the highest binding affinity to the ligand binding pocket
of DHPS of E.coli. As compared to inhibitors i.e Gallic acid and Hippuric acid, antibiotics like Clavalunic acid and
Trimethoprim show higher interactions towards DHPS of E.coli.
Protein binding with various ligands, indicate that various inhibitors of DHFR can be utilized for the treatment of MDR-
UTI after due invivo, invitro and ADMET testing.
Conclusion:
In spite of the enormous success of antibiotics as antibacterial agents, the widespread and uncontrolled use of antibiotics
has led to the emergence of multidrug-resistant (MDR) bacteria (13). Emergence of multidrug resistant (MDR) bacteria
provides with limited treatment options along with increased mortality. Hence, there is an urgent need to search for a
new antibacterial agent. The molecular docking programs aid to establish new ligands/inhibitors for the selected target
receptor proteins from the different available databases, based on their efficiency to bind the active sites on the receptor
(13). Our study shows that Penicillin and clavulanic acid of DHFR and DHPS show higher binding affinity to the
proteins of the folate synthesis pathway of E.coli. These In silico Studies supported with invivo, invitro and ADMET
testing will certainly help towards developing candidates for treatment of MDR-UTI in the future.
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Acknowledgements:
We acknowledge the grant received from R&I Technology Transfer Project funded by RUSA, Maharashtra Government
India for Rs.35 lacs, June 2016, (Sanction No. RUSA/order/R&I/2016-17/273) Dt. 18/6/2016.
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