Discovery studio

Protein structures of HSV-2 gD (PDB. 4MYV), RSAD2 (PDB : 6B4C), and TBK1 (PDB : 4IM0) were collected from PDB database (rcsb.org). Molecule structures of acyclovir (PubChem CID 135398513), quercetin (PubChem CID 5280343), corilagin (PubChem CID 73568), geraniin (PubChem CID 3001497), docosanol (PubChem CID 12620), and MRT67307 (PubChem CID 44464263) were obtained via search from PubChem database (https://pubchem.ncbi.nlm.nih.gov).
Molecular docking was executed for accurate docking of the ligand into the protein active sites using the LibDock module in Discovery Studio (Dassault Systèmes BIOVIA, Discovery Studio Modeling Environment, Release 2017, San Diego: Dassault Systèmes, 2016) [39 (link)]. The interactions were visualized using Discovery Studio Visualizer [40 (link)]. The binding efficiency of each target to the original ligand and prototype compounds was measured using LibDock score [41 (link)]. The LibDock scores were predicted values of the free energy of protein‐ligand binding, and a higher absolute value represents a higher affinity. The most reliable docking pose of each molecule was accepted on the basis of the highest LibDock score and further appraised using Discovery Studio visualizer to examine the molecular interactions.

Molecular modelling of sea bass IgT has been performed by comparative modelling technique. The search for template structures has been performed by means of BLAST (http://www.blast.ncbi.lnm.nih.gov) by selecting the Protein Data Bank archive. The results suggested the structure of mouse IgG as a template. The crystallographic structure is deposited with PDB id code 1IGT [36 (link)]. The 1IGT structure includes four Ig chains: B chain has been used as template for the modelling of 3D structure of sea bass IgT.
Modelling has been performed by using Modeller 9.12 [37 (link)]. The modelling protocol used, as assessed in our lab from previous studies (see [38 (link)] as an example), implies firstly the generation of ten models, and, successively, the selection of the best one in terms of structural quality. Stereochemistry analyses and energy evaluations have been obtained by Vadar web server [39 (link)] and ProsaWeb [40 (link)]. Further structural analyses and visualization have been performed by Discovery Studio (Dassault Systèmes BIOVIA, Discovery Studio Modelling Environment, Release 4.5, San Diego: Dassault Systèmes, 2015).

The H. sapiens HUNK sequence was retrieved from Uniprot (P57058) and used in a protein-protein blast against the Protein Data Bank (PDB). The top scoring PDB structures were retrieved. In addition all protein kinase structure resolved with staurosporine was retrieved from the Protein Data Bank (PDB). A protein sequence alignment was performed between HUNK and the retrieved protein kinases using MUSCLE [29 (link)]. Manual edits were performed using Discovery Studio (Dassault Systèmes BIOVIA, Discovery Studio, Release 2017, San Diego: Dassault Systèmes, 2018). Homology models bound with staurosporine were generated from two template structures (PDB entries: 1NVR and 1WVY) using modeler 9v19 [30 (link)]. Amino acids are numbered with the initiating methionine set to 1. The models were subjected to quality analysis using the PDBsum generator (http://www.ebi.ac.uk/pdbsum) [31 (link)]. The models were prepared for analysis using the protein preparation wizard in which protonation states were assigned followed by an energy minimization to relieve unfavorable constraints (Schrödinger Release 2017-3 Schrödinger, LLC, New York, NY, 2017).

Molecular modeling studies were carried out on a Dell Precision workstation with Intel (R) Xeon (R) CPU E5–1620 v3 @3.50GHz processor. Structure building, docking and analysis were carried out using Discovery Studio (Dassault Systèmes BIOVIA, Discovery Studio Modeling Environment, Release 2017, San Diego: Dassault Systèmes, 2016), GOLD (Genetic Optimization for Ligand Design) suite, version 5.4.0, protein ligand docking package [18 (link)–20 (link)], and the PyMOL Molecular Graphics System, Version 1.8 Schrödinger, LLC.

The development and validation of structure-based pharmacophore models for chymase was made by the Receptor-Ligand Pharmacophore Generation protocol of Discovery Studio (Dassault Systèmes BIOVIA, Discovery Studio Modeling Environment, Release 4.5, San Diego, CA, USA, 2015), using as reference the crystal structure of human chymase complexed to the inhibitor 2-[3-({methyl[1-(2-naphthoyl)piperidin-4-yl]amino}carbonyl)-2-naphthyl]-1-(1- naphthyl)-2-oxoethylphosphonic acid (OHH) from the RCSB Protein Data Bank (PDB) [27 (link)] (PDB code 1T31 [7 (link)]), as described in our previous work [16 (link)]. The 3D structures of 13 active compounds from chamomile extract (alpha-bisabolol, alpha-farnesene, alpha-pinene, bisabolol, caffeic acid, chamazulene, chlorogenic acid, herniarin, matricin, nobilin, patuletin, salicylic acid, and umbelliferone) available from the PubChem database [28 (link)] were downloaded and mapped against the 10 pharmacophore models developed in our previous work [16 (link)], using Discovery Studio to evaluate their matches according to the pharmacophore features identified previously.

The three-dimensional structure of the three different variants of the zinc knuckle domain were built using a threading approach which combines three-dimensional fold recognition by sequence alignment with template crystal structures and model structure refining. The query-template alignment was generated by HHPRED (https://toolkit.tuebingen.mpg.de) and then submitted to the program MODELLER^{84 (link)} as implemented in Discovery Studio (Dassault Systèmes BIOVIA). Query coverage and e-value score were considered to define a suitable template structure. The structure of nucleocapsid protein NCp10 of retrovirus MoMuLV, which contains a single Cys-X2-Cys-X4-His-X4-Cys zinc knuckle domain bound to the oligonucleotide d(ACGCC) was selected as template (https://www.rcsb.org/structure/1a6b). Zinc ions and oligonucleotides were explicitly considered through molecular modeling steps. Fifty models, optimized by a short simulated annealing refinement protocol available in MODELLER, were generated and their consistency was evaluated on the basis of the probability density function violations provided by the program. Stereochemistry of selected models was checked using the program PROCHECK^{85 (link)}. Visualization and manipulation of molecular images were performed with Discovery Studio (Dassault Systèmes BIOVIA).