Evaluación de la Toxicidad Aguda de Extractos de Plantas Medicinales

2.2.10.1. Software Tools

The Schrödinger Suite 2017 is a proprietary software package for studying biomolecular interactions. The various modules and workflows of the suite are accessed through a graphical interface (Maestro). For this work, the applications and workflows listed in Table 2.5 were used. The Schrödinger Suite has been extensively tested and compared to other software tools and its performance is consistently in the top half of the group of tested software tools (Cheng, et al., 2009, Englebienne, et al., 2009, Halgren, et al., 2004, Kellenberger, et al., 2004, Perola, et al., 2004, Repasky, et al., 2007) and often one of the top-ranked tools for docking calculations.

Table 2.5: Schrodinger Suite 2017 applications used in this work.

Application Task

Maestro (version

11.1.012) Graphical interface for all Schrodinger applications and workflows LigPrep Generation of accurate 3D molecular models for ligand

molecules, including tautomeric, stereochemical, and ionisation variation optimised for further computational analyses. These tools are based on well-known bond length, bond angle and atom size parameters and molecular geometry and bonding rules.

Glide An extensive validated ligand placement algorithm and scoring function package to accurately predict binding mode for ligand-receptor complexes.

Prime Protein structure predictions, protein structure refinement and MM-GBSA calculations.

Workflow Task

Protein Preparation Wizard

Correcting common structural problems (e.g. missing residues or residue side chains) and creating reliable, all atom protein models.

Induced Fit

Docking A novel method for fast and accurate prediction of ligand induced conformational changes in receptor active cavities. Combines Glide and Prime to

exhaustively consider possible binding modes and the associated conformational changes within the receptor binding cavity.

The process for docking as implemented in the Schrodinger Suite is illustrated in Figure 2.1. The ensemble of poses obtained as output from this process were further evaluated to obtain information from force field scoring functions.

Download PDB file of protein Delete duplicate protein chains

Delete water molecules not involved in ligand binding Add missing residues (fill gaps) using primary amino acid sequence

Add hydrogen atoms Minimise filled gaps

Crude protein (receptor) structure

Protein Preparation Wizard Align structure in workspace Assign bond orders and formal charges

Fill missing side chains of residues using primary amino acid sequence Add hydrogen atoms

Refine structure and relieve stain and re-orient side chains Prepared protein

(receptor) structure

Docking grid

Build ligand in Maestro

LigPrep Set ionisation state Generate stereoisomers

Generate tautomers Generate ring conformations

Minimise structures Prepared ligand structure Glide dock or Induced -fit dock Ranking and selection of best pose

2.2.10.2. Receptor Model Development

The x-ray crystal structures of the ER LBDs listed in Table 2.2 were used to develop models for docking. Where multiple polypeptide chains were present in the PDB file, chain A was arbitrability selected (because even single polypeptide proteins have chain A) for the experiment. All water molecules except for the one involved in the hydrogen bond bridge with Glu353 and Arg394 in ERa models and Glu305 and Arg346 in ERb models were removed. Protein Preparation Wizard was used to assign bond orders, add hydrogen atoms and fill in missing side chains within the protein structure. The modified protein structure was then energy minimised to optimise bond distances and angles.

2.2.10.3. Ligand Structure Development

For each ligand, LigPrep was used to determine the ionisation state for carboxylic acids and amines at pH 7 ± 2 (this is close to the cellular pH of 7.4); generate tautomers (e.g. keto- enol); generate alternative chiralities for all stereocentres and sample ring conformations to find lowest (i.e. the most thermodynamically stable) energy conformations. Finally, the geometry of each structure was optimised which involved allowing the structure to relax in all dimensions to achieve a low energy conformation. Not all possible isomers were produced because of internal filtering to eliminate structures which violate geometric restrictions such as for fused ring systems or conflict with natural product chiralities such as for the steroid framework. For some ligands LigPrep produced more than one stereoisomer (e.g. 17a- and 17b-estradiol were produced). For those ligands where it was known which stereoisomer was the desired or correct option, only that stereoisomer was used for docking (e.g. only E2 was used). For equol, 2 possible isomers were produced; however, only the S isomer is found in vivo; therefore, the S-isomer was used in the study.

2.2.10.4. Rigid Receptor Docking – RRD

RRD was carried out using Glide and was run within Maestro using the applications menu. The first step in Glide docking was to generate a receptor grid. The LBC binding cavity was defined automatically based on the size, shape and location of the co-crystallised ligand of the particular receptor model used (e.g. 1ERE). For the AF-2 site the centroid of the residues Lys362 and Glu542 in ERa models and Lys314 and Glu493 in ERb models were used to define the location of the binding cavity. The size was determined by specifying ligands to be docked less than 20 Å in length because it must fit the receptor site. The receptor grid was kept rigid – with no van der Waals radius or change scaling of receptor atoms. The ligand

was docked flexibly, allowing nitrogen inversions and ring conformations. No constraints (e.g. required hydrogen bonds or covalent bonds to metal atoms) were specified and all receptor hydroxyl groups were allowed to rotate.

Glide XP was used for docking using default parameters. The core, constraints and similarity options were not used. The core options allow for constraint of ligand poses within the binding cavity based of a reference ligand. The similarity options incorporate a similarity between a docked ligand and a reference ligand – a measure of how alike or unlike the two molecules are. Both the core and similarity options are useful in virtual screening application for drug target development to aid in the identification of strong binding ligands from a pool of potential candidates, which were not applicable to the task at hand. All the ligands listed in Table 3.3 were docked with receptor models listed in Table 3.2. For RRD, poses were ranked by XPGlideScore which was reported in units of kcal/mol and converted to kJ/mol.

2.2.10.5. Induced-fit Receptor Docking - IFD

IFD was run from within Maestro using the workflow menu (Table 2.6). For the LBC, the Glide grid was defined automatically based on the size and orientation of the co-crystallised ligand. The AF-2 site Glide grid was defined using the centroid of the residues Lys362 and Glu542 in ERa models and Lys314 and Glu493 in ERb models estimating the location of the binding cavity and the size was determined by specifying ligands to be docked less than 20 Å in length. Initial Glide docking allows residue side chains to be trimmed (temporarily

removing them from the protein structure) automatically, based on the B-factor. The B-factor is also called the temperature factor and the higher the value, the more mobile the residue side chain. The more mobile side chains were trimmed as their spatial positions were less certain. The parameters used are summarised in Table 2.6. During the induced fit refinement step, the residues that were allowed to adapt to the placement of the ligand were those within 5 Å of the co-crystallised ligand.

For IFD, poses were ranked by IFDScore. The IFDScore is defined as the sum of XPGlideScore and 5% of the Prime energy (i.e. the total energy of the receptor-ligand complex), so this ranking accounts for the overall energy of the complex, not just a

favourable binding energy. The calculated binding energy is given by the scoring function XPGlideScore.

Table 2.6: IFD parameters.

Glide Grid Setup

Box Centre Centroid of ligand

Box Size Automatic based on co-crystallised

ligand for LBC docking, centroid of Lys362/314 and Glu542/493 for AF- 2 docking

Step 1: Initial Glide docking

Protein Preparation constrained

refinement Yes

Trim side chains Yes, automatic based on B-factor

Receptor van der Waals scaling 0.7

Ligand van der Waals scaling 0.5

Number of poses retained per ligand 5

Step 2: Prime induced fit refinement

Refine residues Within 5 Å of ligand

Step 3: Glide re-docking

Glide Docking XP mode; re-dock into structures

within 30 kcal/mol of best structure and within the top 5 structures overall

2.2.10.6. XP GlideScore

XPGlideScore was used in both RRD and IFD to estimate binding energy of the ligand with the receptor. XPGlideScore is optimised to identify and estimate ligand poses that have unfavourable energies based on well-known principles of physical chemistry (e.g.

inappropriate hydrophobic interactions, poor hydrogen bonding interactions, etc.) to weed out false positives. As a result, more precise poses are produced than with standard GlideScore; however, the cost for this increased precision is computational time. Standard GlideScore is used for ligand database enrichment to quickly identify good candidates; however, for a more thorough study XPGlideScore was used.