Download Virtual Screening of Potential Drug-like Inhibitors against MexB Efflux Protein

2011 International Conference on Bioscience, Biochemistry and Bioinformatics
IPCBEE vol.5 (2011) © (2011) IACSIT Press, Singapore
Virtual Screening of Potential Drug-like Inhibitors against MexB Efflux Protein
from Pseudomonas Aeruginosa
Mohanalakshmi N.
Aparna V.
Junior Research Fellow, Dept. of Bioinformatics
School of Bioengineering, SRM University
Kattankulathur, Tamilnadu, India
[email protected]
Junior Research Fellow, Dept. of Bioinformatics
School of Bioengineering, SRM University
Kattankulathur, Tamilnadu, India
[email protected]
Waheeta Hopper
Professor & Head, Dept. of Bioinformatics, School of Bioengineering, SRM University
Kattankulathur, Tamilnadu, India
[email protected]
contributes to both intrinsic and acquired resistance in P.
aeruginosa, while the MexCD-OprJ and MexEF-OprM
efflux system contributes to acquired resistance. The inner
membrane component MexB is responsible for the substrate
specificity and uses proton electrochemical gradient to
transport its substrates and virulence factors that are
important for the colonization and infection of host cells [6].
A widely exploited strategy is the development of inhibitors
of efflux pumps [7], which are intended for combination
therapy with antibiotics. Inhibition of efflux pumps provides
a better way to restore the activity of antibiotics. To date, no
efflux pump inhibitors have been approved for the treatment
of bacterial infections [6], hence this area affords promising
role in drug development. The aim of this study was to
elucidate the mode of binding of substrates to the efflux
pump and also to identify the efflux inhibitory potential of
compounds with structural similarity to the known inhibitor
MC-207,110 using virtual screening and molecular docking
approaches.
Abstract—Prevalence of multidrug resistant Pseudomonas
aeruginosa strains in clinical setup demands the need for
developing potent drugs against efflux mechanisms. In this
study, drug-like molecules with structural similarity to the
known efflux inhibitor were used in order to screen for
potential inhibitors. A total of 167 compounds were screened
and the Glide XP shortlisted compounds were selected based
on Glide score and interaction with active site residues. The
virtual screening method was also applied to study the binding
modes of substrates. The shortlisted compound could have a
better ability to inhibit the MexB protein, hence will provide
adjunctive therapy t o the antibiotics.
Keywords-Pseudomonas aeruginosa;
efflux pump; MC-207, 110; Glide
I.
virtual
screening;
INTRODUCTION
Toxic compound efflux from cells is a general
mechanism that bacteria have developed to protect
themselves against the adverse environments. Efflux pumps
are proteins involved in the transfer of toxic compounds
from the cell which confers the resistance to drug and a wide
variety of structurally unrelated compounds [1].
Pseudomonas aeruginosa is a notoriously difficult organism
to control with antibiotics or disinfectants [2]. The
organism’s intrinsic resistance has been attributed to the
outer membrane, a barrier of limited permeability [3]. While
the reduced uptake likely does limit access of antimicrobials
to their targets within the cells, additional resistance
mechanisms such as drug efflux, helps to reduce the
threshold concentration of antibiotics within the cells [4].
The efflux pumps of P. aeruginosa belong to Resistance
Nodulation Cell Division (RND) family which forms
tripartite system consisting of three components: a
transporter (efflux) protein in the inner membrane (MexB), a
periplasmic accessory protein, (MexA) and an outer
membrane protein (OMP) channel (OprM) [5]. MexABOprM is the first identified tripartite system [6], which
II.
METHODS
A. Receptor X-ray structure
The X-ray crystal structure of the inner membrane
protein MexB (PDB code: 2V50) (Fig.1), in complex with
non-ionic detergent n-dodecyl-D-maltoside resolved at 3.0Å
was downloaded from Protein Data Bank (PDB) [8]. The
asymmetric unit contained two structurally identical trimers,
subunits ABC and DEF. Among the three chains of the
MexB structure the B subunit was considered as the binding
subunit [9].
B. Protein preparation
PDB files of the protein may have various problems that
need to be corrected before proceeding with Virtual
Screening studies hence bond orders, ionization states, steric
clashes and side chain orientations were fixed and energy
minimization was performed with the force field Optimized
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potential for liquid simulations 2005 (OPLS) using protein
preparation wizard of Schrödinger suite version 9 [10]. The
active site of the protein was defined and grid files were
generated using receptor grid generation panel. Grid files
represent physical properties of a volume of the receptor
(active site) that are searched when attempting to dock a
ligand.
bonding mode of the inhibitors and their structurally similar
compounds were compared with those of the substrates.
III.
C. Substrates of MexB
The MexB efflux pumps of P. aeruginosa are of
particular interest because of their exceptionally broad
substrate specificity. The following reporter antibiotics were
used as phenotypic markers of the Mex efflux pumps of
interest: carbenicillin, erythromycin, levofloxacin, Fig.2 [11].
In order to compare the binding modes of the inhibitors with
the substrates, docking was perfumed with the substrates like
carbenicillin, erythromycin, levofloxacin using glide module
of Schrodinger suite v 9.
D. Ligand Database
MC-207,110 (Fig.2) has been identified in Highthroughput screening of small molecule libraries as an
inhibitor of the three multi-drug resistance (MDR) pumps
(MexAB-OprM, MexCD-OprJ and MexEF-OprN) in clinical
isolates of P. aeruginosa [12]. Similarities in structures are
indicative of similarities in bioactivities, therefore, structure
based searching of PubChem database (>85% similarity) was
carried out. This resulted in 167 compounds. The Structure
files of these compounds were obtained from PubChem
Compound Database [13].
The geometries of these compounds were optimized by
adding hydrogens and eliminating unwanted structures using
LigPrep module and Database of chemical compounds was
created using Schrodinger suite.
E. Virtual Screening studies for substrates
The virtual screening of substrate database with prepared
protein was performed with OPLS2001 force field using
virtual screening workflow (VSW) module of the
Schrodinger Suite. VSW uses glide docking to rank the best
compound which utilizes the scoring functions, High
throughput virtual screening (HTVS), standard precision (SP)
and extra precision (XP). HTVS and SP modes are used for
large set of ligands and XP docking is more accurate than the
above two methods. It uses the ligand poses that have a high
score from SP docking. The XP GlideScore scoring function
was used to order the best ranked compounds and the
specific interactions like pi-cation and pi-pi stacking were
analyzed using XP visualizer in Glide module. The binding
mode and interactions of the substrates were ascertained to
compare with the inhibitors.
RESULT AND DISCUSSION
Computer based strategies for structure based drug
discovery presents a valuable alternative to the costly and
time consuming process of random screening. In the present
study, two different virtual screening approaches were used.
First, MC-207,110 analogues were docked into the active site
of MexB protein. For the validation of docking process, the
substrates of the MexB were introduced as a control with the
purpose of screening the compounds with docking score
greater than the substrate.
The docking of the substrates to the active site is shown
in Fig.3a-c, the substrates carbenicillin, erythromycin and
levofloxacin exhibited molecular docking with Glide score
value of -4.271, -3.672, -3.507 and -3.025 (kcal/mol)
respectively. The substrates docked near the vicinity of the
amino acids Thr495, Gly461, Arg468 and Gly97 and were
observed be form hydrogen bond (Table I).
Out of the 167 compounds virtually screened, the
shortlisted
molecule
[(2R)-2,5-diamino-N-[(2S)-3-(4methylphenyl)-1-(naphthalen-2-ylamino)-1-oxopropan-2yl]pentanamide (Pubchem ID: 21970537)] was obtained
from the XP output based on its good interaction, forming
hydrogen bonds with the active site residues Arg468, Ala37,
Gln96 and Glide score of -5.344 (Fig. 4 and Table II).
Hydrogen bonding measures the intermolecular interaction
between the protein and ligands. The compound possessed
1.78 score in the hydrophobic map prediction, hppmap tool
of Schrodinger suite (Fig. 5) proved its strong binding and
interaction. The shortlisted inhibitor docked with better Glide
score than the substrates. However hydrogen bonding with
the active site residue Arg468 was observed dominant among
both the inhibitor and substrate. It is known that MC-207,110
is a competitive efflux inhibitor which competes for the
binding sites in the receptor with the substrates [14].
IV.
CONCLUSION
Combination therapy is a significant approach to combat
drug resistance in bacteria. Efflux pump inhibitors can serve
as an additional drug to alleviate the activity of the existing
antimicrobials. In this study, molecular docking approach
was imparted to study the binding modes and to correlate the
docking score between substrates and inhibitors of efflux
pump MexAB-OprM. The docking studies provide better
insights onto the binding mechanism of substrate and
inhibitor at the molecular level. The virtually screened and
shortlisted compound could act as a potential competitive
inhibitor with the substrate for MexAB-OprM efflux pump.
ACKNOWLEDGMENT
F. Virtual Screening studies for inhibitors
The ligand based virtual screening of inhibitor compound
database with prepared protein was performed with
OPLS2001 force field using virtual screening workflow
(VSW) module of the Schrodinger Suite. The same protocol
used for the substrates was followed. The interactions and
The authors are thankful to Department of Biotechnology
(DBT), Ministry of Science and Technology, Government of
India for their financial assistance and SRM University for
their support.
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TABLE I.
D--H---A
Pubchem ID:
(Arg468)N--H---O
(Ala37) N--H---O
N--H---O (Ala37)
N--H---O (Gln96)
21970537
Distance
(Å)
2.883
3.128
2.891
2.733
Glide Score
(Kcal/mol)
-5.344
Figure 1. Structure of MexB protein from P. aeruginosa
INTERACTION OF ANTIBIOTIC SUBSTRATES OF MEXABOPRM EFFLUX PUMP WITH MEXB PROTEIN
D--H---A
Distance
(Å)
Glide
Score
(Kcal/mol)
Carbenicillin
O--H---O(Thr495)
(Gly461)N--H---O
2.629
2.620
-4.271
Erythromycin
(Arg468)N—H--O
2.877
-3.672
Levofloxacin
(Gly97) N--H---O
3.081
-3.507
Substrates
TABLE II.
Inhibitor
Figure 2. Structure of MC-207,110, an inhibitor of MexB efflux pump
and substrates
INTERACTION OF XP SHORTLISTED COMPOUD (PUBCHEM
ID-21970537) WITH MEXB PROTEIN
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Figure 3. a. Interaction of MexB (B-chain) with carbenicillin (The
substrate is in ball and stick model and the amino acids in lines)
Figure 4. Interaction of MexB (B-chain) with XP docked inhibitor
PubChem ID: 21970537 (in ball and sticks)
Figure 4. b. Interaction of MexB (B-chain) with erythromycin
Figure 5. Hydrophobic map of XP output for the inhibitor showing the
strong hydrophobic patches (blue)
Figure 3. c. Interaction of MexB (B-chain) with levofloxacin
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