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ÁLVARO CORTÉS CABRERA Y CRISTINA RUEDA PÉREZ AN. R. ACAD. NAC. FARM.

2. MATERIALS AND METHODS

2.1. Ligands/Enzyme models

To achieve a reliable model, the structures of the ligands (Table 1)
were energy minimized, in first place using the MM2 force field,
and afterwards with the semi-empirical Hamiltonian AM1 (Austin
Model 1), as implemented in MOPAC 7.1. The structures resulting
from this procedure were used as the starting point of docking assays.

The atomic model of GR from X-ray diffraction studies was
obtained from Brookhaven Protein Data Bank (PDB code: 1M2Z).
Following, the ligand binding domain (residues from 521 to 777)
was extracted discarding all crystallographic water and the ligand
(dexamethasone). To avoid the high energy interactions present in
the crystal structure, a two parts energy minimization protocol was
carried out. In the first part, the structure was minimized by 400
steps of steepest descents (SD), carefully observing the root mean
square deviation (RMSD) from the initial crystallographic positions
to avoid distortion of the structure. In the last part, 1500 steps of
Polak-Ribiere conjugate gradient (GC) were applied with the same
considerations about distortion of the structure. This protocol was
carried out using the GROMOS 96 43a1 force field and the suite
GROMACS 4.0.3.

2.2. Docking

All docking studies were carried out by the program Autodock4 (7)
version 4.0.3 which allows a very fast energy evaluation using
precomputed grids of affinity potentials for rigid docking. In order to
explore the conformational space of the ligands, all torsional bonds in
substrates were set free to perform flexible docking while the enzyme
was kept rigid. Polar hydrogens and Gasteiger charges were assigned
by the respective modules in Autodock Tools (7).

With every ligand, we developed a rigid docking assay with a grid
box of 40 amstrong × 40 amstrong × 40 amstrong placed at the
crystallographic coordinates of the ligand with a spacing of 0.375
amstrong between points, assuring coverage over the active center.

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