The with highest negative CDocker interaction energy, obtained from

The in silico methodology was initiated by docking 64 to the lipoamide binding site and the allosteric site of PDK1 crystal structure (2Q8G).  The X-ray crystal structure of the PDK1 was obtained from the Protein Data Bank (http://rcsb.org/pdb), and the protein was prepared by employing the protocol “prepare protein” of Discovery Studio Client 2017 (DS). This step integrates, correction of the protonation states, the addition of missing residues and minimization of the protein. The ligand structures were subjected to a conjugate gradient minimization for a maximum of 20,000 steps using CHARMm force field.  The docking was performed using CDOCKER module of DS.  The configuration settings were as follows (-17.40, 12.94, -7.03 radii 9.54 for the lipoamide site and 38.06, 32.68, 88.60 radii 7.81 for allosteric site). The best pose, with highest negative CDocker interaction energy, obtained from the docking was taken further for Molecular dynamics (MD) simulation.  Docking studies, when extended to MD, is considered to give a more profound basis for protein-ligand interaction. The protein-ligand complex was solvated with water in an orthorhombic box with an explicit periodic boundary model to simulate the cellular environment. Counter ions were added randomly to neutralize the system. MD simulations were instigated using ‘Standard Dynamics Cascade’ Protocol of DS program.  Initial minimization was carried out using steepest-descent algorithm for 20,000 steps and it was further followed by conjugate gradient minimization for 10,000 steps. Heating was carried out for 400ps where the initial temperature of 50K was raised to 300K at a step size of 2fs. The structure was equilibrated for 1ns and the production run under NVT conditions for additional 1ns. Simulations were extended further for 30ns in an isothermal-isobaric ensemble using NAMD 2.1. The protein-ligand binding site was analyzed using the final structure obtained from the MD. The distance between an active site residue (SER 174 for the allosteric site and PHE 65 for lipoamide site) and the protein was obtained from the last 10ns of the simulation.