Computational Studies of N-1 Substituted Quinolone Derivatives as Potent Inhibitors of Gyrb Subunit of Escherichia Coli K-12

Introduction

It has been observed that quinolone compounds are powerful antibacterial agents¹. Based on this ,various quinolone analogues have been discovered till today with improved bactericidal activity against both gram positive and gram negative bacteria^2-3. Day by day increasing resistance power of bacteria ,challenges the  medicinal chemists to design and develop  new more  powerful antibacterial drugs. These drugs targets on bacterial DNA gyrase and topoisomerase IV⁴ as they have structural and functional similarity. DNA gyrase and topoisomerase IV consist of two pairs of  subunits : A2B2 (GyrA , GyrB) and C2E2 (ParC , ParE) respectively. GyrA and ParC subunits breaks and rejoins DNA strands thereby releasing torsional stress. On the other hand, GyrB and ParE provide energy to the enzyme by ATP hydrolysis. Quinolones works as  GyrA inhibitors by forming DNA-enzyme complex, which resist the re-joining of broken DNA strands. However,  due to its wide clinical use, side-effects and bacterial resistance have emerged. There is a need to research on designing and developing inhibitors having different mechanism of action.

Since 1960, GyrB inhibitors have not been in clinical use due to their consequential side-effects⁵. Computational methods⁶ are being used to analyse both unbound and bound structures which help in designing relevant structures useful for biological activity. Molecular docking is a competent tool in studying various interactions between inhibitor molecules and active sites of the target receptor molecules⁷.  Here in our work we have studied the binding interactions of 2a,b with GyrB subunit of Escherichia coli K-12 (PDB code : 6YD9) and compared the antibacterial activity obtained during our study^8-9.We have evaluated binding affinities, root mean square deviation (rmsd), inhibition constant (Ki) and visualized different interactions such as hydrogen bonding, pi-anion, pi-alkyl, pi-cation, salt bridges in 3D as well as 2D.

Materials and Methods

Synthetic approach

We have previously synthesized 4-oxo-1,4-dihydro-3-[1-oxo-2-hydrazino-3-{p-toluenesulfon}]quinoline N-R (R = H and Et)  2a,b synthesizedfrom N-R (R = H and Et) substituted 1,4-dihydro-4-oxoquinoline-3-carbohydrazide 1a,b (Scheme 1) in our laboratory^8-9and evaluated their antibacterial activity.

Scheme 1 Click here to View Scheme

Docking study

Molecular docking is a key tool used for structural analysis and computer aided drug design (CADD)¹⁰. It helps in predicting the binding modes of a ligand with a receptor molecule in three

dimensional structure¹¹. All the experiments were conducted using default parameters to get accurate results. We have performed docking with softwares Autodock4 (AD₄) and Vina¹². The 3D and 2D visualization of the complex was assisted with Discovery Studio Visualizer software. The 3D structures of our molecules  2a and 2b  is shown in Figure 1.

Figure 1: 4-oxo-1,4-dihydro-3-[1-oxo-2-hydrazino-3-{p-toluenesulfon}]quinoline 2a; C17H15N3O4S ; M.M. = 357.3837. 1-Ethyl-4-oxo-1,4-dihydro-3-[1-oxo-2-hydrazino-3-{p-toluenesulfon}]quinoline 2b; C19H19N3O4S ; M.M. = 385.4336 Click here to View figure

Preparation of Ligand and Receptor

GyrB subunit of Escherichia coli K-12 was selected as the receptor molecule which was obtained from Protein Data Bank file (PDB : 6YD9). The water molecules and other heteroatoms bound to the receptor were removed, followed by addition of polar hydrogen atoms. The receptor molecule was further refined by checking missing atoms, adding Kollmann charges and completing the incomplete residues¹³. Autodock tools 1.5.7^14-15 were used to parameterize the rigidity of receptor and flexibility of ligands which were then saved in PDBQT file. Here Gasteiger-Marsili method, was used to estimate the charges¹⁶.

Molecular Docking via AD₄

After the preparation of receptor, a grid box of size 100 x 100 x 100 ( x, y and z) points and spacing 0.375Å was generated using Autogrid4. The number of genetic algorithm (GA) was set to 10 with population size of 150. The GA number of evaluations were set to 2500000 which corresponds to medium option. Best conformers were searched by choosing Lamarckian genetic algorithm. AutoDock analyzer was used to investigate the results. The complex was visualized by using Discovery Studio visualizer for better interpretation. We have used standard docking protocol and the final result is reported for the conformer having lowest binding energy. 

Molecular Docking via AutoDock Vina

Docking by Vina was conducted with same grid size as AD₄. The exhaustiveness was set to 8 (short option) and  maximum energy range between the best and worst docking modes were set to 4 kcal/mol respectively. Docking was performed by using configuration file containing grid box properties and ligand-receptor information. The results were generated in a log file showing positional binding affinity (kcal/mol) along with root-mean-square deviation values. The conformer with lowest binding affinity was selected for the final results.

Results and Discussion

Docking results using AutoDock  (AD4)

Molecular docking was performed by following standard protocols. Summary of the results is listed in Table 1. Compound 2b showed good activity ‘in vitro’ against E. coli (ETEC)⁹   and gave binding energy of -7.81 kcal/mol. It shows the occurrence of one hydrogen bonding of oxygen of SO₂ with Thr A:165 having bond length 1.92Å (Table 2). Other interactions such as salt bridge formation, pi-alkyl, pi-sigma are shown in Figure 2. Compound 2a showed a binding energy of -8.19 kcal/mol, showing the occurrence of one hydrogen bond with NH of  hydrazide and Val A:43 having bond length 2.11 Å  (Figure 2).

Docking results using AutoDock Vina

Compounds with lowest binding affinity is chosen as the best model for interpretation. Compounds 2a and 2b showed binding affinity of -5.9 and -5.7kcal/mol respectively by using AutoDock Vina software (Table 1). Compound 2a formed one hydrogen bond with Lys A:189 having bond length 2.62 Å. Compound 2b formed one hydrogen bond with Arg A:190 having bond length 2.03 Å (Table 3). Other interactions such as pi-sigma, pi-alkyl, pi-cation, and pi-anion are also shown in Figure 3.

Table 1 : Result Analysis by both AutoDock 4 and AutoDock Vina

Result Analysis	Receptor	Compound	Docking score	Amino acid residues
AutoDock 4	6YD9	2a	-8.19	ALA A: 47, ASN A : 46, GLU A : 50, ILE A:78, THR A:165, VAL A:43, VAL A: 71, VAL A:167
		2b	-7.81	ALA A:47, ASP A:73,  GLU A:50, ILE A:78, ILE A:94, PRO A:79, VAL A:120, VAL A:167, THR A: 165
AutoDock Vina	6YD9	2a	-5.9	ARG A : 190, LYS A:189, THR A:34
		2b	-5.7	HIS A:38, ARG A:190, GLU A:193

 

Table 2 : Docking results using AutoDock 4

Ligand	Binding Energy (kcal/mol)	Inhibition constant, Ki	No. of H-bonds	H-bond length (Å)	Amino acids
2a	-8.19	986.68 µM	1	2.11	ASN A: 46
2b	-7.81	1.88 µM	1	1.92	THR A:165

 

Table 3 : Docking results using AutoDock Vina

Ligand	Binding Affinity (kcal/mol)	No. of H-bonds	H-bond length (Å)	Amino acids
2a	-5.9	1	2.62	LYS A :189
2b	-5.7	1	2.03	ARG A:190

 

Figure 2 : Docking by AD4 software ; Images : Discovery Studio Visualizer ; (i) 2D image of 2a (ii) 3D image of 2a (iii) 2D image of 2b (iv) 3D image of 2b Click here to View figure

Figure 3 : Docking by Vina software ; Images : Discovery Studio Visualizer ; (i) 2D image of 2a (ii) 3D image of 2a (iii) 2D image of 2b (iv) 3D image of 2b Click here to View figure

Conclusion

In this study we have explored the protein-ligand interactions of our synthesized quinolone derivatives 2a and 2b. Both AD4 and Vina are popular tools in studying protein-ligand interactions. GyrB subunit of Escherichia coli K-12 was chosen for this study. Both the compounds were successfully docked with the receptor molecule.  The best docking pose with lowest binding affinity was explored for protein-ligand interactions. The comparative study showed that  both the moieties formed hydrogen bond at the active site. It can be inferred that the compounds can show antibacterial property. According to the docking results, both moieties have comparable binding affinities but 2b formed shorter hydrogen bond (bond length = 1.92Å and 2.03 Å) with amino acid residues in comparison to 2a (bond length = 2.11Å and 2.62 Å). Compound 2b also showed significant antibacterial activity in vitro as compared to 2a.

Acknowledgement

This work was financially supported by World Bank Sponsored MHRD project TEQIP III, for Seed grant [File No.: MJPRU/TEQIP 3/MRPS/01-Nivedita Srivastava, Dt 15/06/2019]. We also acknowledge Center for Computational Research and Biology, Scripps Research for developing programs ‘AutoDock4’ and ‘AutoDock Vina’ for docking studies.

Conflict of Interest

There is no conflict of interest.

Funding Sources

There is no funding Source.

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