INSILICO MODELING AND DOCKING STUDIES OF NEW DELHI METALLO BETA LACTAMASE-1 (SUPER BUG)

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INSILICO MODELING AND DOCKING

STUDIES OF NEW DELHI METALLO

BETA LACTAMASE-1 (SUPER BUG)

*DR. JAYASREE GANUGAPATI

Department of Biotechnology

Sreenidhi Institute of Science and Technology (SNIST) (affiliated to Jawaharlal Nehru Technological University),

Yamnampet, Ghatkesar, Hyderabad Andhra Pradesh, India, Pincode-501301

SIRISHA V. MUKKAVALLI

SAHITHI ATIMAMULA

DR.K.S.R.SIVA SAI

Abstract

New Delhi Metallo Beta Lactamase-1 (NDM-1) is a novel beta -lactamase enzyme that is ubiquitously found in Escherichia coli. This enzyme belongs to a B1 subclass of Metallo Beta Lactamases and is known to induce resistance to standard intravenous antibiotics. The tertiary structure of NDM-1 was predicted using Modeller9v7, based on the structural homology of the x-ray crystallographic structures of VIM-2 & VIM- 4 (Verona imipenemase-2 & -4) proteins from Pseudomonas aeruginosa. Further refinement of the structure was done using the loop modeling. Docking Analysis of NDM-1 with flavonoids was then performed using, GOLD, Auto Dock and Argus Lab. The analysis of the results of all three docking softwares suggested that Quercetin may be a potential inhibitor of NDM-1. Further analysis in the wet lab may provide us more information regarding inhibiting of NDM-1.

Keywords: NDM-1, Homology modeling, Quercetin.

Introduction

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carbapenem Klebsiella pneumonia [Rolain, 2010]. These Beta lactamases can be classified based on function into four groups(1,2,3,4) and based on molecules (nucleotides and amino acid)into 4 groups (A,B,C,D). The NDM-1 enzyme belongs to the class B1 of metallo-beta lactamase (MBL) which can hydrolyze penicillin, cephalosporin and carbapenems [Yong ,et al.,2009; Queenan ,et al., 2007]. All of these enzymes are characterized by their ability to induce resistance to standard intravenous antibiotics prominently used for the treatment of severe infections.

This resistance is a result of the proteins ability to hydrolyze ß-lactam antibiotics,and closely related proteins that have no ß-lactamaseactivity. The hydrolysis causes the inactivation of the ß-lactam antibiotics by hydrolyzing the ß-lactambond in those drugs. Originally this resistance to ß-lactam antibiotics was restricted geographically and limited only to specific bacterial species. These species specific barrier was overcome because of the location of the gene coding for this protein being located as mobile genes on plasmids that can readily spread through bacterial populations. The geographical restriction was overcome due to globalization, allowing an international dissemination of such bacteria. [Queenan, et al., 2007; Rolain, 2010] The gene coding for NDM-1 is now becoming a common variant in the bacterial genome, and is currently found in an about 1-3 percent of enterobacteria infections.

The growing increase in the rates of antibiotic resistance is a major cause for concern in isolates of the Enterobacteriaceae family. The absence of any effective antibiotics for the treatment of NDM-1 positive patients restricts containment of this gene to only being brought about by good infection control in hospitals. Lack of information regarding the structure is a major drawback for finding effective antibiotics for the treatment of patients. Hence an attempt was made to predict the three dimensional structure of NDM-1, which was further used for carrying out docking studies.

Flavonoids are a group of polyphenolic compounds that are ubiquitous in nature and are categorized, according to chemical structure, into flavonols, flavones, flavanones, isoflavones, catechins, anthocyanidins and chalcones. These flavonoids have several potential beneficial effects on human health, such as antiviral, anti-allergic, antiplatelet, anti-inflammatory, anti-tumor and antimicrobial activity. As a result of its antimicrobial effects, we hypothesized that they may have potential effects in inhibiting the action of NDM-1. For our studies the flavonoids present in green tea, a rich source of dietary flavonoids, were chosen for carrying out the docking studies. The information about the flavonoids present in green tea was obtained from USDA Database for the Flavonoids Content of Selected Foods (March 2003). (Table 1)

Table 1 : List of flavonoids& their classification found in green tea ( obtained from USDA Database )

Materials and Methods

The amino acid sequences of New Delhi Metallo Beta Lactamase (158 amino acids) of Escherichia coli (GenBank ID: 300422616) was obtained from National Center for Biotechnological Information (NCBI).

S.NO FLAVAN-3-OLS FLAVONES FLAVONOLS

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1. Homology Modeling

1.1 Template selection for NDM-1

BLASTP [Altschul et al., 1990] search was then performed against PDB in NCBI database, by submitting the amino acid sequence in FASTA format, in order to identify suitable templates with known structures. Vim-2 (PDB ID-1KO3) and Vim-4 (PDB ID- 2WHG) proteins were identified as suitable templates bearing high percentage( 45%) similarity to the query sequence .

Multiple Sequence Alignment (MSA) is used to align more than two sequences thereby representing the occurrence of one or more patterns common to a set of sequences, regions of variations and the nature of the mutations/substitutions. CLUSTALW is an online tool used for the construction of MSA. The hits obtained by performing BLASTP were aligned with query (NDM-1) using CLUSTALW [Higgins et al., 1988] and EXPRESSO (3DCoffee).

An optimal alignment between the target sequence (NDM-1) and templates (VIM-2, VIM-4) was obtained. (Figure 1).

Figure 1: Conserved residues coordinating zinc ions are denoted with yellow color. Additional amino acids unique to NDM-1 are enclosed

in a purple box.NDM-1 also has a unique HXHXD motif among the mobile MBLs indicated by red box.

1.2 Model Construction:

Modeller9v7 is a program that is used for homology or comparative modeling of the 3-D structure of proteins and their assemblies by satisfying the spatial restraints.

Hundred 3-D models of NDM-1 were generated based on alignment with two templates VIM-2 and VIM-4 at a resolution of 1.90°A.

1.3 Model Screening

The models predicted by the above mentioned approach were screened using SAVES server to identify the best possible models. Models with relatively low energy and 0.8% in the disallowed regions were selected for carrying out further refinement. RAMPAGE analysis of the selected models showed that residue, 30: THR, was lying in the outlier region.

1.4. Model Refinement

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1.5 Energy Refinement and Model Evaluation:

The model’s energy computations were done with the GROMOS96 implementation of Swiss-PDB Viewer [Van et al., 1996]. RMSD, a good indicator of uncertainty in the atomic coordinates, [Laskowski., 2003] was calculated for the model with VIM-2 and Vim-4. The model obtained was found to be of high stereo chemical quality as interpreted from PROCHECK analysis considering parameters like covalent bond distances, angles [Laskowski et al. 1996].

2. Docking Studies

2.1 Preparation of Ligands:

Based on the Anti-microbial activity, Flavonoids, were considered as possible inhibitors of the NDM-1 protein. The CID files of the Ligands were then downloaded from PUBCHEM. The minimization of the prepared Ligand was carried out with the GROMOS96 implementation of Swiss-PDB Viewer [Van et al., 1996]. Mol Inspiration, an online tool, enables us to perform QSAR studies in order to identify potential inhibitors to biological targets. Lipinski’s Rule of Five was then applied to select probable ligands.

2.2 Active Site Analysis:

Q-site Finder, an online tool which uses hydrophobic probes, was used to predict possible binding sites. Energetically favorable probes sites were clustered and then ranked according to the sum of interaction energies. (Figure 2)

Figure 2: The blue color region indicates the most probable Active Site

2.3 Docking Studies:

To study the nature of interactions, binding mode and selectivity of NDM-1 protein with individual flavonoids, docking was carried out with CCDC GOLD., Autodock 4.0 and Arguslab 4.0.1.

2.3.1 Genetic Optimization for Ligand Docking (GOLD)

GOLD is a commercial Package available for docking studies which uses Genetic Algorithm (GA) to understand the flexibility of the Ligand and selected receptor hydrogen’s. [Jones et al.,1997]

The GOLD Score was calculated by defining the site using the list of atom numbers and retaining all the other default parameters.

2.3.2 Autodock 4.0

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The energy grid was built within a cubic box of dimensions 40X16X50 A° with a spacing of 1.0 A°. The docking was performed based on Lamarckian Genetic Algorithm.

2.3.3. Arguslab 4.0.1

Arguslab 4.0.1 is Molecular modeling and Drug Docking software. It is very flexible and can reproduce crystallographic binding orientations. Arguslab, which provides a user friendly graphical interface and uses ShapeDock algorithm, was used to carry out docking studies of NDM-1.[ Thompson et al]

Results:

The 3D model that was obtained using multiple templates (VIM-2 & VIM- 4) by the above mentioned approach was visualized (Figure. 3). Superimposition of the final model with VIM-2 and VIM-4 (Figure. 4) resulted in RMSD values of 0.66 and 1.27 A° respectively thereby reinforcing the reliability of the templates The stereo chemical quality of the model was assessed using RAMACHANDRAN PLOT analysis (Figure. 5), where 83.8% of the residues were in the most favored region, 15.4 % in allowed region, 0.8 % in generously allowed region and 0% of the residues lying in the disallowed regions.

Further studies using PDBSUM and Verify 3-D were also carried out to check the reliability of the model. The plot indicates that none of the residues lie in the disallowed region. The model thus obtained in this study is the best model of NDM-1. The 3-D structure of NDM-1 protein was then used to predict possible inhibitors by carrying out docking studies with green tea flavonoids.

The Mol Inspiration data of the compounds was then analyzed using Lipinski’s Rule of Five. Those compounds that had more than one violation (i.e.thearubigins, theaflavins and its derivatives, Epicatechin-3-gallate, Epigallocatechin, and Epigallocatechin-3-gallate) were eliminated.

Q-site finder predicted 10 different sites. The first site was considered the most probable binding site. The residues that formed the binding site in NDM-1 were identified as ALA-10,140;ASN-12,156;ASP-60;CYS- 144;GLN-59;GLY-13,142,143;HIS-56,58,125;ILE-146;LEU-154;LYS-61,147;MET-90;PHE-6,141;SER-11;TRP-29;VAL-9 .This includes the Histidine residues important for zinc binding.

The compounds were then docked using each of the three docking softwares. The fitness score from GOLD and the energy values from the three docking softwares are indicated in Table 2. The binding energies obtained in Arguslab ranged from 7.10 to 12.61 kJ/mol & Autodock yielded energies ranging from 3.83 to -17.20 kJ/mol.

The results of CCDC GOLD can be analyzed both in terms of energy values, which ranged from -4.80 to -31.78 kJ/mol, as well as fitness scores, ranging from 27.67 to 41.03.

The results indicate that quercetin is the best inhibitor.The interaction of quercetin with NDM-1 is indicated in Figure 6.

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Figure 4: White color indicates NDM-1. Vim-4 and VIM-2 are indicated by Red and Green color respectively.

Figure 5 : PROCHECK ANALYSIS

Table 2: Energy and fitness score values of the docked ligands

S.NO

NAME OF THE FLAVANOID

Arguslab Autodock GOLD DATA

Energy Values Energy Values Energy

Values

Fitness Score

1 Quercetin -9.14522 -14.86 -18.16 41.03

2 Luteolin -7.82065 -14.55 -16.03 40.42

3 Kaempferol -8.46425 -14.33 -14.63 40.32

4 Apigenin -8.61544 -14.57 -13.72 38.91

5 Caffeine -4.87115 -10.88 -4.80 35.63

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Figure 6: NDM-1 – Quercetin complex: Pink represents binding site residues; Yellow color represents the ligand; Grey color represents the rest of the protein

Discussion:

The aim of the study was to identify the most accurate model for NDM-1 and a potent inhibitor of the same. The homology modeling of NDM-1 using multiple templates yielded a final model with least energy and no residues in disallowed region of the Ramachandran plot. The final refined model was then used for docking studies with green tea flavonoids.. Docking results of all the three docking softwares indicated quercetin as a potent inhibitor of NDM-1.Further analysis can be carried out in the wet lab.

Acknowledgements:

We would also like to thank the management of Sreenidhi Institute of Science and Technology (SNIST) for their encouragement and support in carrying out the work.

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