Pymol 0.99rc6

All tested compounds were prepared in LigPrep (Schrödinger) and exported to mol2 files. Docking studies were performed with GOLD 5.3 (CCDC). Before docking, BACE1 protein (PDB code 4D8C) was prepared using Hermes 1.7 (CCDC). All histidine residues were protonated at Nε, the hydrogen atoms were added, a ligand and water molecules were removed. The binding sites were defined as all amino acid residues within a radius of 15 Å from ligand (BXD) in the BACE1 complex. We applied the automatic settings of the genetic algorithm for very flexible ligands (number of operations equal 125,000). We received 10 conformations by the ligand sorted by the GoldScore scoring function. Results of docking were visualised with PyMOL 0.99rc6 (DeLano Scientific LLC, San Francisco, CA).

The nucleotide sequence of EpTrx was obtained from the complete genome sequence data58 (link), and its amino acid sequence was analyzed using DNAMAN. The three dimensional structure of Trx was predicted using SWISS-MODEL (http://swissmodel.expasy.org/) and software PyMOL 0.99rc6 (DeLano Scientific LLC, USA). The 3D structure of Trx from Saccharomyces cerevisiae (PDB number 2HSY) was used as a structural template. Sequences of Trx from other organisms were downloaded from Genbank for comparison (Supplementary Table S1).

Corina online (Molecular Networks and Altamira) was used to create three-dimensional structures of compounds, that were then prepared using Sybyl 8.0 (Tripos). Protonation states were inspected, hydrogen atoms were added, atom types were checked and Gesteiger-Marsili charges were assigned. All ligands were docked to acetylcholinesterase from 2CKM and to butyrylcholinesterase based on a 1P0I crystal structure using GoldSuite 5.1 (CCDC). Before docking, the proteins were prepared in the following way: all histidine residues were protonated at Nε, the hydrogen atoms were added, ligand and water molecules were removed; the binding site was defined as all amino acid residues within 10 Å from bis-(7)-tacrine for AChE, and 20 Å from the glycerol molecule for BuChE. A standard set of genetic algorithms with a population size of 100, number of operations 100 000, and clustering with a tolerance of 1 Å was applied. After docking process, 10 ligand poses, sorted by GoldScore (for AChE) and ChemScore (for BuChE) were obtained. The results were visualised by PyMOL 0.99rc6 (DeLano Scientific LLC).

Protein was determined (Bradford, 1976 (link)) with a commercial dye reagent (from Bio-Rad) using bovine serum albumin as standard. However, with S. elongatus extracts, to avoid chlorophyll interference, protein was determined according to Lowry (Lowry et al., 1951 (link)), again with a commercial Lowry reagent (Modified Lowry protein Assay Kit, from Thermo Scientific) and bovine serum albumin as standard, measuring the absorbance at 750 nm.
Protein structures were represented using PyMOL 0.99rc6 (DeLano Scientific LLC), utilizing the Protein DataBank (PDB; http://www.rcsb.org/pdb/) files 2XG8, 2XKO, and 2V5H for PII-PipX, NtcA-PipX, and PII-NAGK, respectively (Llácer et al., 2007 (link), 2010 (link)).

The three-dimensional structures for ligands were prepared with LigPrep (Schrodinger Suite) from SMILES strings, using an ionizer for the prediction of protonation states at pH 7.4 ± 0.2. The previously prepared homology model of the histamine H₃ receptor was utilized for docking studies [32 (link)]. This model was built with Modeller 9.14 based on a template of M₃ muscarinic receptor (PDB code: 4U15) using automodel class and very fast refinement option. Recently, the crystal structure of H₃R was obtained [38 (link)]. Comparing this experimental structure with our homology model, it is worth noting that the model is very consistent with the X-ray structure. Therefore, the application of our homology model in presented docking studies was fully justified. All dockings were performed using the Induced Fit Docking procedure (Schrodinger Suite) with the standard protocol. Binding site box with a center at Asp3.32 and size, adjusted for docking of ligands with length ≤25 Å, was applied. The obtained ligand–receptor complexes were analyzed concerning IFDScore as well as interaction networks with Maestro (Schrodinger Suite) and PyMOL 0.99rc6 (DeLano Scientific LLC).

The 3-dimentional structure of synthesized compounds were drawn in Corina on-line (Molecular Networks and Altamira) [38 ] and subsequently prepared using Sybyl 8.0 (Tripos, Certara, Princeton, NJ, USA). [39 ] Atom types were checked, hydrogen atoms were added and Gasteiger–Marsili charges were assigned. Acetylcholinesterase from the 2CKM and butyrylcholinesterase from the 1P0I crystal structures were prepared before docking in the following way: histidine residues were protonated at Nε, the hydrogen atoms were added, water molecules and ligands were removed. Docking was performed with GoldSuite 5.1 (CCDC). [40 ] We defined binding sites as all amino acid residues within a radius of 10 Å from bis-(7)-tacrine for AChE and 20Å from the glycerol molecule for BuChE. The standard settings of the genetic algorithm with a population size 100, number of operations 100,000 and clustering tolerance of 1 Å was applied. As a result, we obtained 10 ligand poses, sorted by GoldScore (for AChE) and ChemScore (for BuChE) function value. To visualize the results, we used PyMol 0.99rc6 (DeLano Scientific LLC, Schrodinger, Cambridge, MA, USA) [41 ].

Ligand
structures were prepared with LigPrep (Schrodinger Suite) from SMILES
strings. Protonation states were predicted with Epik at pH 7.4 ±
0.2. The recently obtained crystal structure of H₃R was
used for the studies (PDB code: 7F61).^{58 (link)} The binding
modes of the analyzed compounds were predicted by the Induced Fit
Docking procedure (Schrodinger Suite) based on the standard protocol
and the OPLS_2005 force field. The binding site was defined as a box
with the center at Asp3.32 and size adjusted to allow docking of ligands
with length ≤25 Å. The obtained ligand poses were assessed
by IFDScore and GlideScore. The interaction networks were visualized
with Maestro (Schrodinger Suite) and PyMOL 0.99rc6 (DeLano Scientific
LLC).