Journal of Science and Technology Ubon Ratchathani University : Special Issue November 2017

Structure based drug design of 4-aminoquinilone derivatives in DNA Gyrase B subunit for anti-tuberculosis agents using molecular dynamics simulations

Naruedon Phusi1, Chayanin Hanwarinroj1, Kampanart Chayajaras1, Nitima Suttipanta2, Pharit Kamsri3, Auradee Punkvang3, Khomson Suttisintong4, Patchareenart Saparpakorn5, Supa Hannongbua5, Prasat Kittakoop6, James Spencer7, Adrian Mulholland8 and Pornpan Pungpo*1

1 Department of Chemistry, Faculty of Science, Ubon Ratchathani University,85 Sthollmark Rd., Warinchamrap, Ubonratchathani, 34190, 2 Faculty of Pharmaceutical Science, Ubon Ratchathani University, 85 Sthollmark Rd., Warinchamrap, Ubonratchathani, 34190, Thailand 3 Faculty of Science, University, Nakhon Phanom, 48000, Thailand 4 National Nanotechnology Center, 114 Thailand Science Park, Pathumthani, Thailand 5 Department of Chemistry, Faculty of Science, , Chatuchak, Bangkok, 10900, Thailand 6 Chulabhorn Research Institute and Chulabhorn Graduate Institute, Vipavadee-Rangsit Highway, Bangkok, 10210, Thailand 7 School of Cellular and Molecular Medicine, Biomedical Sciences Building, University of Bristol, Bristol, BS8 1TD, United Kingdom 8 Centre for Computational Chemistry, School of Chemistry, University of Bristol, Bristol, BS8 1TS, United Kingdom *E-mail: [email protected]

Abstract Mycobacterial DNA gyrase B subunit, GyrB has been identified to be one of the potentially underexploited drug targets in the field of antitubercular drug discovery. 4-aminoquinoline derivatives were developed as potential GyrB inhibitors. In the present study, we applied molecular dynamics simulations to elucidate the binding mode and the key interactions of 4-aminoquinoline derivatives in the GyrB pocket. The obtained results indicated that the crucial interactions of the highest active compound were hydrogen bond interactions with Met93(A) and electrostatic interactions of carboxylic group with Arg185(B) sidechain in the GyrB pocket. This interaction absented in other compounds. Moreover, hydrophobic interactions are also important for binding affinities in GyrB binding site of 4-aminoquinoline derivatives. Therefore, the obtained results from this study provide informative structural concept for designing of novel 4-aminoquinoline derivatives with better potency of GyrB inhibitors against Mycobacterial tuberculosis.

Keywords : 4-Aminoquinoline derivatives, GyrB, anti-TB agents, molecular dynamics simulations

Introduction The clinical efficacy of fluoroquinolone drugs Tuberculosis ( TB) , caused by Mycobacterium demonstrated over the past 20-30 years has validated tuberculosis (M. tuberculosis) , remains a major global DNA gyrase as a target in the area of broad-spectrum health problem. In 2015, an estimated 9.6 million people antibacterials [ 2] . Gyrase A subunit, GyrA has been developed TB and 1.8 million died from the disease[1]. facing a major hurdle of their resistance developed by

31 Journal of Science and Technology Ubon Ratchathani University : Special Issue November 2017

M. tuberculosis which makes gyrase B subunit a drug able target for discovery of potent anti- tuberculosis agents. DNA gyrase B subunit, GyrB is involved in the process of ATP hydrolysis which in turn provides energy Figure 1 The structures of 4-aminoquinoline derivatives to gyrase A subunit for maintaining the DNA topological (a) compound 1 (IC50 7.89 μM) and (b) state[3]. So, GyrB has been genetically demonstrated to compound 2 be a bactericidal drug target in M. tuberculosis, but there (IC50 11.33 μM) have not been any effective therapeutics developed The biological activities of these compounds against this target for TB [4]. Recently, 4-aminoquinoline were expressed in terms of MsmGyrB assay ( IC in derivatives have been developed as anti-tuberculosis 50 μM) values. The chemical structures of these inhibitors agents with moderate biological activities against M. were constructed using the standard tools available in tuberculosis and XDR M. tuberculosis [ 5] . These GaussView 3.07 program and were then fully optimized compounds were tested against MsmGyrB with using the M062X/ 6- 31G* * method implemented in moderate activities based on ATPase assay and DNA Gaussian 09 program. The initial coordinates for supercoiling assay. To develop these derivatives as molecular dynamics simulations of the complexes was highly potent GyrB inhibitors, the key interactions for obtained from molecular docking calculations using binding of 4- aminoquinoline derivatives in GyrB Autodock 4.02 program. GyrB structure for docking was binding site are required. Therefore, molecular downloaded from protein databank (PDB Code: 4B6C). dynamics simulations approaches have been used to Molecular dynamics simulations were performed to understand dynamic behaviour, the binding mode and predict the inhibitors in the GyrB binding pocket. TIP3P key binding interactions of these derivatives. The water model and Na+ were chosen to represent water obtained results derived from this work aid to rational for salvation and ions for neutralize system. To reduce design of new potential GyrB inhibitors as anti- the bad steric interactions of solvate water molecules tuberculosis agents. + and Na ions of each system, the inhibitor-GyrB complex Propose approach was first minimized by 1,000 steps with atomic positions Two inhibitors, the highest active compound 1 and the of solute species restraint with using force constant of lowest active compound 2 (Figure 1) were selected to 500 kcal/mol Å2. Non-bonded cut-off was set to 8 Å. The model the binding mode and key binding interactions in threshold value of the energy- gradient foe the this work. The chemical structures and their convergence was set as 0.001 kcal/mol/ Å. Then, the experimental biological activities of 4- aminoquinoline whole system was minimized by 1,500 steps as the derivatives were selected from the literature [5]. same conditions of water and ions minimization without restraining condition. Next, the systems were gradually warmed up from 0 to 300 K in the first 20 ps followed by maintaining the temperature at 300 K in the last 10 ps with 2 fs time simulation steps in a constant volume

32 Journal of Science and Technology Ubon Ratchathani University : Special Issue November 2017

boundary. The solute species were restrained to their based on the equilibrium state obtained. The binding initial coordinate structures with a weak force constant free energies were calculated to evaluate the binding of 10 kcal/mol Å2 during the temperature warming. This affinities of inhibitors in GyrB binding pocket using the was followed by 70 ps of the position- restrained Molecular Mechanics Poisson-Boltzmann Surface Area dynamics simulation with a restrain weight of 2 kcal/mol (MM-PBSA) [6-9] and Normal-mode [10] methods. 10 Å2 at 300 K under an isobaric condition. Finally, 15 ns snapshots were used to calculate the binding free molecular dynamics simulations without any restraints energy in this work. The single snapshot that showed were performed using the same conditions. The root- the calculated binding free energy closed to mean square deviations (RMSDs) of the GyrB enzyme experimental binding free energy was selected to and the inhibitors, binding interactions were analysed analyze the binding mode and binding interactions.

Figure 2. RMSD plots of (a) compounds 1 and (b) compound 2 complexed with GyrB

Figure 3. Binding mode of (a) compound 1 and (b) compound 2 in GyrB binding pocket derived from molecular

dynamics simulations.

Table 1. The calculated binding free energies (kcal/mol) of 4-aminoquinoline inhibitors calculated by MM-PBSA method. Energy contribution (kcal/mol) Cpd. ΔEMM ΔGpolar ΔGnon ΔGsolv H -TS Gcal. Gexp. 1 -51.93 19.83 -48.61 23.15 -28.78 13.46 -15.30 -8.07 2 -48.51 19.87 -42.72 25.66 -22.85 13.30 -9.55 -7.01

33 Journal of Science and Technology Ubon Ratchathani University : Special Issue November 2017

RESULTS AND DISCUSSION hydrogen bond interaction of carboxylic group of Structural stability during MD compound 2 with amide sidechain of Asn45(A) of DNA simulations and binding free energy calculations gyrase B subunit. Moreover, hydrophobic interactions of In order to determine structural stability during this compound with Val42(A), Val70(A), Val92(A), molecular dynamics simulations of the highest active Ile164(A) sidechains were observed. To gain quantitative compound 1 and less active compound 2 in GyrB binding insights into the affinity for binding of 4-aminoquinoline pocket, the RMSDs for all atoms of solute species inhibitors in the GyrB binding site, the binding free relative to the initial structure over the 15 ns of simulation energies of 4- aminoquinoline/ GyrB complexed were times were calculated and plotted in Figure 2. The calculated by the MM-PBSA method as shown in Table plateau characteristic of the RMSD plot over the 1. The obtained results showed that the calculated simulation time is the criteria to indicate the equilibrium binding free energy of compound 1 and compound 2 are state of each solute species. Convergent RMSD plots -15.32 kcal/mol and -9.55 kcal/mol, respectively. The indicate that the equilibrium state of the highest active obtained result indicated that binding free energy (Cpd.1) and the less active (Cpd.2) compound was calculations based on MM- PBSA method produced reached after 12 ns. The obtained results from the last good value and closed to experimental binding free 3 ns were mainly analysed in more details of binding free energy of compound 1 and compound 2, -8.07 kcal/mol energy and binding interactions. and -7.01 kcal/mol respectively. It is notable that the Binding mode and binding interactions of calculated free binding energies of inhibitor are in the compounds correct order as compared with the IC50 (experimented The crucial interactions of compounds, the binding free energy) values. The obtained results could highest active compound 1, with amino acids be successfully used to validate the molecular dynamics surrounding the binding pocket of GyrB enzyme was simulations procedure in this study. The obtained results analysed as shown in Figure 3 ( a) . The major in this work are reliable. interactions in the GyrB binding pocket is electrostatic Conclusion interaction between carboxylic group (COOH) to nitrogen Molecular dynamics simulations were atom of Arg185(B) sidechain of DNA gyrase B subunit. successfully applied to model the reliable binding Hydrogen bond interactions of trifluoromethyl with NH modes, inhibitor-enzyme interactions and binding free sidechain of Met93(A) was obtained. Moreover, energies of 4-aminoquinoline derivatives in the GyrB hydrophobic interactions with Val42(A), Val70(A) and binding pocket. Hydrogen bond, electrostatic and Val93(A) sidechain of DNA gyrase B were found as an hydrophilic interactions play the important role for additional contribution for binding in GyrB binding site. binding of 4-aminoquinoline derivatives. Accordingly, The binding mode of the less active compound 2 bound the key structural for binding and dynamical concepts with GyrB pocket observed from the molecular dynamics are fruitful to design new potential GyrB inhibitors simulations are shown in Figure 3 ( b) . The major against M. tuberculosis. interaction in the GyrB binding pocket is the crucial

34 Journal of Science and Technology Ubon Ratchathani University : Special Issue November 2017

Acknowledgments Evaluation" . European Journal of This research was supported by the Medicinal Chemistry. 103: 1-16. Thailand Research Fund (RSA5980057 and [6] Homeyer, N.; Gohlke, H., 2012. "Free Energy MRG5980152), the National Research Council of Calculations by the Molecular Mechanics Thailand, Higher Education Research Promotion and Poisson-Boltzmann Surface Area Method". the Center of Excellence for Innovation in Chemistry Molecular Informatics. 31: 114-122. (PERCH-CIC). Faculty of Science, Ubon Ratchathani [7] Hou, T. et al., 2011. "Assessing the Performance University, University of Bristol and NECTEC are of the MM/PBSA and MM/GBSA Methods. gratefully acknowledged. 1. The Accuracy of Binding Free Energy Calculations Based on Molecular Dynamics References Simulations" . Journal of Chemical [1] Organization, W. H. 2015. Global Tuberculosis Information and Modeling. 51: 69-82. Report. [8] Wang, J. et al., 2006. "Recent Advances in Free http://www.who.int/tb/publications/global_re Energy Calculations with a Combination of port/en/ (accessed 26 October). Molecular Mechanics and Continuum [ 2] Maxwell, A. , DNA Gyrase as a Drug Target. Models". Current Computer-Aided Drug Portland Design. 2: 287-306. Press Limited: 1999. [9] Wang, J. et al., 2001. "Use of Mm-Pbsa in [3] Lewis, R. J. et al., 1996. "The Nature of Inhibition Reproducing the Binding Free Energies to of DNA Gyrase by the Coumarins and the Hiv-1 Rt of Tibo Derivatives and Predicting Cyclothialidines Revealed by X- Ray the Binding Mode to HIV-1 RT of Efavirenz Crystallography". The EMBO Journal. 15: by Docking and MM-PBSA". Journal of the 1412-1420. American Chemical Society. 123: 5221- [4] Kaur, P. et al., 2009. "Delineating Bacteriostatic 5230. and Bactericidal Targets in Mycobacteria [10] Kaledin, M. et al., 2004. "Normal Mode Analysis Using Iptg Inducible Antisense Expression". Using the Driven Molecular Dynamics PLoS ONE. 4: e5923. Method. Ii. An Application to Biological [5] Medapi, B. et al., 2015. "4-Aminoquinoline Macromolecules" . The Journal of Derivatives as Novel Mycobacterium Chemical Physics. 121: 5646-5653. Tuberculosis Gyrb Inhibitors: Structural

Optimization, Synthesis and Biological

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