Computational protocol: A structural insight into the negative effects of opioids in analgesia by modulating the TLR4 signaling: An in silico approach

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Protocol publication

[…] Although the association between TLR4/MD2 and MD2 with the morphine, M3G, and naloxone is well established, no co-crystal structures are available to date. The structures for TLR4/MD2 (ID 3FXI, co-crystalized with LPS), MD2 (ID 2E59, co-crystalized with lipid IVa), and TLR4/MD2 (ID 5IJB, without ligand), were retrieved from the protein data bank (PBD) and used in docking studies and as controls. These structures were checked for abnormalities and then prepared for docking simulations with morphine, M3G and naloxone. There are 3 states of MD2, based on the position of the Phe126 sidechain in the gating loop, that have been reported in crystallographic studies. In the closed state (MD2C) Phe126 is bent towards the hydrophobic cavity of MD2 where it binds to an agonist, in the open state (MD2°) the Phe126 sidechain is exposed to solvent and an antagonist is bound, and in the Apo state (MD2apo) MD2 is not bound to any ligand, and the sidechain of Phe126 is reported to be exposed to solvent (). Our docking simulations were performed exclusively with the MD2C state, in the presence and absence of bound TLR4. MD2°-lipid IVa, MD2C-LPS, and MD2apo were used as controls for comparative dynamics studies with MD2C-naloxone, MD2C-morphine, and MD2C-M3G. MD2apo was considered as a control for comparative analysis of the shrinkage in MD2 cavity, however, we did not perform detail simulation of this state as this has been previously investigated.The structures for morphine, M3G, and naloxone were retrieved from the PubChem database, prepared for docking using the MOE suite of tools, and then docked with either the MD2C monomer or the heterodimeric TLR4/MD2C complex. Ligands were docked with MD2C using three different tools; MOE, AutoDock 4.2, and Patchdock. MD2 displays one dominant hydrophobic pocket that guides the most favorable docking conformation, and all of the tools produced similar docking results with exceptionally low RMSD of the MD2-ligand complexes (). Therefore, the top scoring poses for each ligand provided by MOE were selected for further analysis. A detailed description of the docking protocol has been discussed previously. [...] GROMACS 5.0.7, along with the recently released CHARMM36 (C36) all-atom additive protein force field (ff) module, recommended for the simulation of protein structures,was used for the simulation of docked complexes and selected controls. As the ligand atoms are not recognized by the C36 ff module, the parameters for morphine, M3G, and naloxone were prepared using the SwissParam server, and their charges were calculated in the MOE suite (2015.1) using the MMFF94 ff module. All simulated systems were solvated in a dodecahedron box, using the TIP3P water model, and neutralized with the addition of counter ions. Conditions mimicking a physiological salt solution were created, with Na and Cl ions being added to a concentration of 0.1 M.The solvated systems were then energy-minimized using a steepest decent algorithm to remove unwanted steric clashes from all protein-ligand complexes. After energy minimization, the systems were grouped into protein-ligand and solvent-ions to avoid collapse, and then equilibrated for 1 ns in a constant volume (NVT). A constant temperature of 300 K was achieved using a Berendsen thermostat algorithm. All systems were equilibrated for a further 1 ns at a constant pressure (NPT) of 1 bar using a Berendsen barostat. All short-range non-bonded interactions were calculated within a cut-off of 1.2 nm, and long-range electrostatic interactions were computed using the particle mesh Ewald method with a cut-off of 1.2 nm. The LINCS algorithm was employed to constrain all bonds, and an integration time-step of 2 fs was allowed for all systems. All simulations were performed using the NPT ensemble for a total of 120 ns, and coordinates were saved at 2 fs intervals. [...] Most of the data were analyzed with built-in modules of GROMACS v5.0.7. Graphical images were produced with PyMol and VMD. Interface analyses were performed in MOE, VMD, and UCSF Chimera, and images were generated in MOE and Chimera. All computational studies were performed on a Dell PowerEdge server with a CentOS6 GNU/Linux operating system. […]

Pipeline specifications

Software tools GROMACS, SwissParam, PyMOL, VMD, UCSF Chimera
Applications Drug design, Protein structure analysis
Chemicals Morphine, Naloxone