An analysis of properties of the binding pocket can suggest features to include in a
pharmacophore query. In the following, a pocket analysis is first demonstrated, then the results of the analysis applied to refining the simple query that was generated in earlier.
Analyzing the Binding Pocket
The binding pocket can viewed both in 2D and 3D to help identify important points of
interaction. A 2D depiction of the ligand interactions with the pocket can be obtained using the Ligand Interactions application. With $MOE/sample/mol/1hpv.pdb loaded in the system, open the panel using
MOE | Compute | Ligand Interactions
In the Ligand Interactions panel, press Isolate to isolate the ligand and pocket residues in the MOE Window. Turn on hydrogen bond display with
MOE | Footer | Contacts
A bound water HOH_201 (also bound to ILE50) and an H-bond interaction of the ligand hydroxyl group with residue ASP25 can be observed in both the Ligand Interactions panel and the MOE Window.
Water HOH_201 bound to ILE50 and H-bond to ASP25
Now we will look more closely at the ligand-pocket interactions in 3D.
Protonated ligand and receptor 1. Protonate the system.
For accurate drawing of surface interaction maps and of creation of volume constraints, explicit atoms are required. Currently, the system is in hydrogen-suppressed mode. The system can be protonated using
MOE | Compute | Protonate 3D
In general, explicit hydrogen atoms are required for all-atom molecular mechanics, dynamics, and electrostatic calculations. The Protonate 3D application assigns ionization states and calculates hydrogen positions in a macromolecular structure.
In the Protonate 3D panel, press OK. The calculation may take a few moments to complete; progress is reported in the MOE Window.
2.
Ligand and receptor, hydrogens hidden
Hide non-polar hydrogens.
Hiding the non-polar hydrogens permits a clearer view of the binding site:
MOE | RHS | Hydrogens
Press the Hydrogens buttons successively until the hydrogen display is as desired.
Successive presses cycle between no hydrogens displayed, polar hydrogens only, and all hydrogens.
Closer examination of the binding site will reveal that the water hydrogens are well-positioned for hydrogen-bonding as is the ligand hydroxyl hydrogen. The ASP25 residue is now protonated as well, and donates an H-bond to the ligand hydroxyl group.
In this image, hydrogen bonds are displayed as white dotted lines, whilst red and purple dotted lines represent ligand-solvent and ligand-pocket interactions respectively, as calculated by the Ligand Interactions application.
3. Draw the pocket.
The binding pocket can be depicted using a 3D molecular surface. Select MOE | Compute | Surfaces and Maps
Molecular surface of pocket, top and side views
Press Apply to calculate the Molecular Surface around the receptor atoms near the ligand. Here, Color has been set to Constant, with the Surface Color orange and the surface type Solid.
By rotating the view to obtain a side view, the active site channel between the A and B chains of the receptor can be seen.
In these images, the transparency of the surface has been adjusted (using the TF and TB sliders in the Surfaces and Maps panel) for better ligand visibility.
4.
Calculate pocket electrostatics.
The electrostatic maps of the 1HPV pocket predict where hydrophobic, hydrogen bond acceptor and hydrogen bond donor contacts are preferred. Partial charges must be
assigned to the system before the calculation is performed. Here, the charges have already been assigned by the Protonate 3D application.
In the Surfaces and Maps panel, change the Surface to Electrostatic Map. Press Apply.
The calculation solves the Poisson-Boltzmann equation for the electrostatic potential, which is then used to generate the acceptor, donor, and hydrophobic predictive maps. The maps show the locations in space at which an atom of the given type has a potential equal to the value (in kcal/mol) given by the corresponding Level slider.
We will now examine each of the electrostatic maps in turn.
5.
Hydrophobic map
Analyze the hydrophobic map.
Isolate the hydrophobic map. The hydrophobic map gives the locations of receptor contact and of low preference for acceptors and donors.
Here, the rendering mode has been set to Solid and the color to green. Hide the other two maps by setting the rendering mode of Acc and Don to None.
There are four -3 kcal/mol hydrophobic regions. The 1HPV receptor comprises two chains identical in sequence, and, accordingly, the hydrophobic areas appear as a
mirrored pair, with one smaller and one larger hydrophobic preference region associated with each chain. These regions are suggestive of locations for hydrophobic and/or aromatic pharmacophore features.
6.
Acceptor map
Analyze the hydrogen acceptor map.
Isolate the hydrogen acceptor map by setting the Hyd and Don rendering modes to None.
Here, the rendering mode of Acc has been set to Solid.
There is a -2 kcal/mol hydrogen acceptor region contacting the bound water HOH_201.
This suggests adding a projected feature to the pharmacophore to contact this area.
7. Analyze the hydrogen donor map.
Donor map
Isolate the hydrogen donor map by setting the Hyd and Acc rendering modes to None.
Here, the rendering mode of Don has been set to Solid.
There is a -2 kcal/mol hydrogen donor region at ASP25 contacting the hydroxyl group on the ligand. This suggests a possible donor pharmacophore feature.
Refining the Query
Having analyzed the ligand-pocket interactions, we can now refine the 1HPV-query1
pharmacophore query by adding the pharmacophore features suggested by the analysis. If it is still open, close the Ligand Interactions panel as it is no longer needed.
1.
Hydrophobic map and query
Open the query.
Open the simple pharmacophore query created earlier:
MOE | File | Open | 1HPV-query1.ph4.
The Pharmacophore Query Editor will open with the 1HPV-query1.ph4 query loaded. At the same time, the query will be rendered in the MOE Window.
2. Tidy up the rendering area.
For better visualization:
o Hide all electrostatic maps by setting their rendering mode to None in the Surfaces and Maps panel.
o Hide the receptor atoms with MOE | Popup | Hide | Receptor.
3.
Acceptor map with query
Add features suggested by the hydrophobic map.
Show the hydrophobic map (in isolation) by setting the Hyd rendering mode to Solid.
The hydrophobic features already in the query can be seen to coincide with the regions of preferred hydrophobic contact. Thus, no additional features are needed to capture the interactions depicted by the hydrophobic map.
4. Add features suggested by the hydrogen acceptor map.
Hide the hydrophobic map (set its rendering mode to None in the Surfaces and Maps panel).
In the Pharmacophore Query Editor, press the Scheme: Info button to open the information panel for the current scheme (Unified). In the Unified Info panel, turn on
Acc2 to enable the projected acceptor annotation points (the pharmacophore type is Acc2) and then close the Info panel. Turn on Show Projecting Vectors in the Pharmacophore Query Editor.
In the MOE Window, projected Acc2 acceptor annotation points will be displayed. It can be seen that the projections from the C=O and SO2 groups coincide with the bound water.
Select these two annotation points, then press Create: Feature. An Acc2 projected feature will be created at the centroid of the selected annotation points.
5.
Donor map with query
Add features suggested by the hydrogen donor map.
In the Unified Info panel, turn off Acc2. In the Pharmacophore Query Editor, change the Ligand Annotation render mode to Long.
In the MOE Window, an annotation point can be seen on the hydroxyl group. Select this annotation point (it is easiest to do this by left-dragging the mouse), then press Create:
Feature to create a Don&Acc feature on the hydroxyl group.
6.
Excluded volumes
Add excluded volumes on the receptor atoms of the binding site to avoid steric clashes within the pocket.
First ensure no atoms are selected using MOE | Popup | Select | Clear. Then, select the binding pocket atoms with MOE | Popup | Select | Pocket.
In the Pharmacophore Query Editor, press Create: Union. This will create a grouped volume feature made up of volumes placed on all selected atoms. By default, the volume is already of type Excluded.
Increase the radius R of the volume feature to 2.0. As explained above in the section on constraints, atoms are considered to have zero radii when evaluating them against volume constraints. To avoid clashes, the volume radius should be increased to take into account the van der Waals radii of the ligand atoms of interest. The value of 2.0 accounts for the atom radii, with some tolerance.
For easier visualization, you can turn off the display of the volumes in the MOE Window by selecting V1 in the feature list of the Pharmacophore Query Editor and then turning on the Hidden checkbox.
When the volume feature is selected in the list and the Hidden attribute is on, the centers of the volumes are marked with selection markers and a dotted contour line drawn around the volumes showing the volume's location.
Note that the Hidden attribute only affects rendering in the MOE Window. It has no effect on searching: hidden features participate in pharmacophore searching as normal.
7. Save the refined query.
Press Save in the Pharmacophore Query Editor. In the Write Query File panel, enter the name 1HPV-query2.ph4 and press OK.