Computational Drug Discovery and Design

1. To set up a DMD experiment structures of the receptor and the
   ligand in one of the standard molecular formats are required. In
   addition, the data about the receptor binding region is needed
   to define the targeted region for the ligand in this local docking
   approach (seeNote 8). Such data can be obtained from muta-
   genesis, mass spectroscopy, NMR experiments, etc.


2. Ligand is placed at a distance of the receptor so that (1) the
   distance between the closest atoms in the receptor and ligand is
   several times higher than the cutoff for the nonbonded inter-
   actions in the used MD simulations (seeNote 9); (2) the short-
   est pathway from the ligand to the receptor is roughly directed
   into the geometrical center of the predefined binding region on
   the receptor surface. The structure of the ligand should be
   previously minimized and can be put in a different random
   orientation for an individual DMD run (seeNote 10).


3. One of the central atoms of the ligand is chosen as aligand
   central atom L.


4. One of the atoms in the core of the receptor is chosen asa core
   atom C(seeNote 11). The vector connectingLandCdefine
   the focus pointFon the surface (Fig.2a).


5. The system then is read into Leap module of AMBER [17],
   standard counter ions and an octahedral solvent box are added
   with the minimum distance from solute atoms to the box
   boundaries of 4 A ̊(seeNote 12). Standard protein force fields
   as FF99SB for parameterization of the protein part of the
   system and GLYCAM 06 [18] for the GAG part are used.


6. An equilibration of the system is carried out, which includes
   minimization of the solvent with the restrained solute
   (10 kcal/(mol A ̊^2 )), minimization of the solute, short
   (10–50 ps) heating of the system to 300 K in NVT canonical
   ensemble with Langevin thermostat with the restraints on the
   solvent. Then, 500 ps MD are carried out with Langevin
   thermostat and Berendsen barostat in NPT ensemble. In all
   MD simulations, the integration step was chosen as 2 fs,
   SHAKE algorithm for bonds including hydrogen atoms and
   Particle Mesh Ewald methods (with the cutoff of 8 A ̊) are used.
   Dihedral angles NMR restraints (10 kcal/(mol A ̊^2 )) are applied
   to GAG sugar rings to keep them in required conformations
   (as suggested by literature or experimental data) because suffi-
   cient sampling of ring conformations is not achieved at the time
   scales of the MD simulations used in this protocol [4].


7. The distances betweenL,F, andCshould be calculated after
   the minimization and MD equilibration steps. The changing
   with the time NMR distance restraint is defined as ||LC|| at
   initial moment of targeted MD,t¼0, and as ||FC|| at the
   final moment of targeted MD,t¼ttMD(Fig.2b). The strength

Solvent Inclusion in Docking Glycosaminoglycans 449