DSV, Vector method

The major advantage of this method is the exclusion of the slow Cerius2 step and the generation of the pharmacophore within DSV. The step-wise detail of the methodology used for the generation of pharmacophore with a ligand in the active site is given below. Equally, water molecules could be used as sources of hydrogen bond donors or acceptors in the absence of ligand. During the course of optimization care was taken in satisfying the overall matching of the desired hydrogen bond in protein ligand interactions. Complementarity between the ligand and protein interface in terms of spatial occupancy and absence of any unfavorable interactions conferring steric conflict between the ligand and the binding pocket of the protein were also dealt with caution .

3.5.2.1 Preparing the PDB file

1) Generate hydrogens for the pdb file both for docking and for pharmacophore searching. This is best done using the What If Web Interface. http://swift.cmbi.ru.nl/servers/html/index.html

Figure 3.6 Image of web interface for what if program

1) Select from options in main window Prepare PDB file for docking programs

2) Prepare PDB file window allows to upload a PDB (Figure 3.7), obtained from the PDB database

Figure 3.7 Image for what if weblink for uploading PDB file

3) Once uploaded a PDB the program will take a few minutes to get to the following page (Figure 3.8).

Figure 3.8 Image of job done result on what if weblink

4) Selecting the results page (Figure 3.9) a file predock.pdb can either be viewed by clicking on the link or saved by saving the link to a file. Rename the file to something sensible e.g. in the case of 2UYN.pdb the file was renamed as 2uynH.pdb

Figure 3.9 what if weblink page image for viewing or saving the .pdb file

3.5.2.2 Generating a pharmacophore search model

1) Use Discovery Studio Visualizer (DSV) by starting the program up on the computer (Figure 3.10).

Figure 3.10 Start up image for DSV, used for viewing .pdb file

2) Select File from the top menu and open the saved PDB file from the respective directory (Figure 3.11).

3) A ligand in the active site helps define the pharmacophore. Either way, centre on the active site and select an atom which is roughly at its centre.

a. Select View from the main menu and then Hierarchy, this shows the individual chains and enables to locate the ligand, selecting the ligand in the side menu highlights the ligand structure in the graphics window.

b. Click on the icon in the menu bar to get centered view on the ligand in the active site (Figure 3.12).

Figure 3.12 Selection of ligand in the binding site of the protein (the selected ligand is highlighted with yellow dots)

c. If there is no ligand in the binding site, use the graphics window to identify a water atom in the active site cavity and click on the icon for a centered view.

4) To generate the exclusion spheres in a radius of about 10Å around the centre of the active site (Figure 3.13).

a. Hide the hydrogens at this stage by selecting Chemistry then Hydrogens then hide from the main menu.

b. Hide the ligand atoms by selecting from the main menu Scripts then Visualizations then Show/Hide Ligands.

c. To select atoms for exclusion spheres select Edit from the main menu then Select.

d. Choose Radius and specify 10 for the number of Angstroms and Apply and then Close.

e. Check by rotating the graphics window that all amino acids around the active site cavity are selected. The value of 10Å could be made larger or smaller to suit the protein in question.

Figure 3.13 Image of window for selection of radius around the binding site in DSV

f. Incase of a mistake use the Ctrl+Z command (as with Microsoft word) to reverse the last action.

g. Unselect the ligand otherwise exclusion spheres will generate for the ligand too! Do this by holding down the Ctrl key and clicking with the left mouse button on the ligand highlighted in the Hierarchy window. h. With protein atoms still selected (highlighted with yellow dots) select

Structure from the main menu, then Query Features, change the feature from centroid to ExclusionSpheres (Figure 3.14) in the menu and click ok

Figure 3.14 Image of DSV with the query feature window

i. This generates a large number of football-like spheres for the pharmacophore (Figure 3.15). The exclusion spheres are not helpful for visualization so hide them by selecting View from the main menu then Visibility in the sub menu and then selecting Hide.

Figure 3.15 Exclusion spheres (highlighted in yellow) around the active site of the protein

3.5.2.3 Generation of hydrogen bond acceptor query feature

1) To generate H-bond acceptor feature unhide the ligand (either using the Hierarchy window or the Scripts option on the main menu). Incase of no ligand water molecules can be used to generate H-bond acceptor features.

2) Select an atom to be a hydrogen bond acceptor on the ligand, typically an oxygen atom (Figure 3.16) and then select Structure from the main menu, and then Query Features and then Acceptor.

Figure 3.16 Introduction of H-bond acceptor vector from the ligand atom towards amino acid residue of the protein

3) The acceptor vector highlighted in yellow is pointing into space and not towards the sensible H-bond donor atom. To change this click the right mouse button on the H-bond acceptor vector and select Attributes of Acceptor at the bottom of the menu (Figure 3.17).

Figure 3.17 Selection window for changing the properties of H-bond acceptor vector in DSV.

4) The first thing to change in the Acceptor feature Attributes window is the Orientation of the vector. Click on the value in the window and it becomes a selectable menu for applying the changes (Figure 3.18). The default is always Projection.

Figure 3.18 Multi options for changing the properties of acceptor feature

5) In this case the Second Lone Pair gives a sensible vector to the arginine in the diagram. Change the Length from 3Å to more sensible value of 2.75Å (Figure 3.19).

Figure 3.19 Changing the length of the H-bond acceptor vector fits it properly towards the arginine of the protein

6) As long as the vector is within a small fraction of an angstrom from the correct position it should be fine. The XYZ values can be changed but this refers to the Acceptor atom position which in this case is the ligand therefore no need to change it.

7) Same procedure can be used for another acceptor atom by repeating steps 1-6. 2-3 vectors are reasonable for the first pharmacophore.

8) The addition of a Location restraint is carried out by selecting the Acceptor Head points in the graphics window or in the Hierarchy window then selecting Structure on the main menu then Query Features and then from that menu select Location (Figure 3.20).

Figure 3.20 Selection of arrow head of H-bond acceptor vector for generating the location restraint

9) Due to a bug in the program the radius value for location sphere defaults to 1.5Å. To change this select the Location spheres individually and change the Radius value manually by hand to 0.8Å for the Acceptor head Location and 0.35Å for the Acceptor tail Location (Figure 3.21).

Figure 3.21 Changing the radius of location sphere around acceptor head and tail

3.5.2.4 Generation of hydrogen bond donor query features

1) The H-bond donor feature is generated in the same manner as for H-bond acceptor (section 3.5.2.3) except instead of selecting a hydrogen bond acceptor atom on the ligand, a donor atom such as hydroxyl oxygen or amide nitrogen is chosen.

2) The only problem is that the donor hydrogen atom may not point in the correct orientation; instead the Donor Head Point could be pointing into space (Figure 3.22). This is a similar problem as for hydroxyl groups as an acceptor point.

Figure 3.22 H-bond donor from a ligand atom (the donor head is pointing in to the space)

3) It is possible to rotate the donor vector around and hence position it close to a suitable hydrogen bond donor atom. Select the middle of the bond between the carbon and oxygen from which the HB Donor vector originates. Use the torsion option from the main menu to rotate around the bond and move the donor tail atom to a reasonable position (Figure 3.23).

Figure 3.23 Optimization of the position of the head of H-bond donor vector by using the torsion icon in DSV

4) After optimizing the position of H-bond donor vector add Location restraints as described in section 3.5.2.3 (8,9)

3.5.2.5 Saving the pharmacophore in .chm file format

1) By making all the H-bond vector features visible along with the exclusion spheres, the pharmacophore file can be saved by selecting File from the main menu and then save as option (Figure 3.24).

Figure 3.24 Saving the pharmacophore file in .chm file format by using the file, save as option of the main menu of DSV

2) To search the generated pharmacophore file against the catalyst, the query file needs to be in .chm format. Select a suitable name and use the extension .chm from the Files of type option.