Computational protocol: In Silico Investigation of Potential TRAF6 Inhibitor from Traditional Chinese Medicine against Cancers

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Protocol publication

[…] The X-ray crystallography structure of the human TNF receptor-associated factor-6 (TRAF6) downloaded from RCSB Protein Data Bank with PDB ID 3HCT [] was employed for virtual screening. PONDR-Fit [] protocol was employed with the sequence of TRAF6 protein from Swiss-Prot (UniProtKB: Q9Y4K3) to predict the disordered amino acids. In preparation section, Prepare Protein module in Discovery Studio 2.5 (DS 2.5) was employed to protonate the X-ray crystallography structure of TRAF6 protein with Chemistry at HARvard Macromolecular Mechanics (CHARMM) force field [] and remove crystal water. Prepare Ligand module in DS 2.5 was employed to protonate the final structure of TCM compounds from TCM [email protected] [] and filter TCM compounds using Lipinski's Rule of Five []. The binding site for virtual screening was defined closed to key residues Glu69, Pro71, Ile72, Leu74, Met75, Ala101, and Pro106. [...] LigandFit protocol [] in DS 2.5 was employed using a shape filter and Monte-Carlo ligand conformation generation to dock the TCM compounds into the binding site. The protocol optionally minimized the docking poses with CHARMM force field [] and filtered the similar poses using the clustering algorithm. Dock Score energy function was employed to evaluate the docking poses using the following equation: (1)Dock Score=−(ligand receptor interaction energy  + ligand internal energy). [...] Gromacs 4.5.5 [] was employed for the molecular dynamics (MD) simulation using classical molecular dynamics theory, which simulates each protein-ligand complex under dynamic conditions. In preparation section, the pdb2gmx protocol of Gromacs and SwissParam program [] were employed to provide topology and parameters for TRAF6 proteins and each ligand, respectively. For solvation with TIP3P water model, the Gromacs protocol defined a cubic box upon the edge approximately 1.2 nm from the protein complexes periphery and creates a neutral system using 0.145 M NaCl model. A maximum of 5,000 steps of steepest descents [] minimization was employed to remove bad van der Waals contacts. In equilibration section, a position-restrained molecular dynamics with the linear constraint algorithm for all bonds was performed using NVT equilibration, Berendsen weak thermal coupling method, and Particle Mesh Ewald method. A total of 10 ns production simulations were performed with time step in unit of 2 fs under NPT ensembles and Particle Mesh Ewald (PME) option. The 10 ns MD trajectories were then analyzed by a series of Gromacs protocols, and the presumable pathways for small molecule under dynamic conditions were analyzed by the CAVER 3.0 program []. […]

Pipeline specifications

Software tools PONDR-FIT, CHARMM, PHENIX, GROMACS, SwissParam, CAVER
Databases UniProt UniProtKB
Organisms Bos taurus
Diseases Hypoxia, Neoplasms, Neural Tube Defects, Substance-Related Disorders
Chemicals Diiodotyrosine