Computational protocol: PknB remains an essential and a conserved target for drug development in susceptible and MDR strains of M. Tuberculosis

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Protocol publication

[…] Based on sequencing of the pknB gene, the sequence that showed mutation was taken for bioinformatics analysis to access the impact of the change. Molecular dynamics simulations (MD) tool have potential to provide detailed information on the fluctuations and conformational changes of proteins and hence was used here. MD-simulations were performed to the docked complex to evaluate the stability, conformational changes and getting insights into the natural dynamic in solutions. MD-simulations were performed using GROMACS version-4.6.4 [–], with the AMBER99SB force field []. The ligand was parameterized by applying the standard RESP procedure using Antechamber [], where charges for free ligand were derived from HF/6-31G* calculation in Gaussian03 []. Histidine residues were assumed to be charged, with the ND and NE atoms protonated; Arginine and Lysine residues were assumed to be protonated. All systems were solvated using explicit TIP3P water model [], in a cubic box with a margin of 10 Å and neutralized by adding Sodium counter-ions. The Particle-Mesh Ewald method was applied to calculate long-range electrostatic interactions with a cutoff distance range of 10 Å, [] and a Lennard-Jones 6–12 potential was used to evaluate van-der-Waals interactions within the cut-off range of 10 Å. The LINCS algorithm of fourth order expansion was used to constrain bond lengths []. After solvation and neutralization, each system was energy minimized for 10,000 steps using steepest-descent optimization method to remove poor van-der-Waals contacts in the initial geometry. After minimization, two stages of equilibration were conducted. First, the system was equilibrated for 100 ps with position restraints of 10,000 kJ/mol on all heavy atoms. A constant temperature of 300 K was maintained using the V-rescale algorithm [], with a coupling time of 0.1 ps and separate baths for the solute and the solvent. Second, the pressure was kept constant at 1 bar using the Parrinello–Rahman pressure coupling scheme with a time constant of 2 ps []. Initial velocities were generated randomly using a Maxwell–Boltzmann distribution corresponding to 300 K. Neighbor lists were updated every 10 fs using a group cut-off scheme. Finally, the production run was performed for 100 ns without restraints at 300 K in the isothermal-isobaric ensemble.Interaction analysis was performed using g_dist tool in the GROMACS package to measure the inter-atomic distances. The g_rms tool of the GROMACS package was used to calculate the root mean square deviations (RMSD) during the trajectories taking the minimized crystal structure as a reference. Root mean square fluctuations (RMSF) of the backbone of each residue were calculated by g_rmsf while g_mmpbsa tool is used to calculate the binding energy. […]

Pipeline specifications

Software tools GROMACS, P-LINCS, g_mmpbsa
Application Drug design
Organisms Corynebacteriales
Diseases Tuberculosis
Chemicals Amino Acids, Ethambutol, Isoniazid, Rifampin, Streptomycin, Ofloxacin