1. Module 2: Structure Based Ph4 Design
MOE: Pharmacophore Derivation and Searching
Module 2: Structure Based Ph4 Design
MOE provides several applications to analyze protein information in absence of ligands:
The Site Finder (using the binding site of a receptor to generate the query)
Contact preferences
In part b) of the SBDD course:
The MCSS algorithm for de-novo design
Docking
Procedure for receptor based design:
Open the protein structure and identify the binding pocket
Use alpha site technology to locate binding site
Generate dummy atoms to mark site
Generate the relevant pharmacophore features based on SAR
Define the general features based biological/chemical knowledge of interactions
Identify key features based on interactions using Contact Statistics or Molecular Surfaces Save pharmacophore model
Refine using Excluded volumes
Search conformational database(s) for ligands that contain the relevant pharmacophores
Re-design the pharmacophore model, if necessary and search again
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2. MOE: Pharmacophore Derivation and Searching
Alpha Site Finder
Objective
The Site Finder screens the surface of a protein for potential binding sites. In addition to locating cavities it also indicates preferred locations for hydrophilic or non-hydrophilic interaction points.
Methodology
Site Finder considers the relative positions and
accessibility of the receptor atoms along with a
rough classification of chemical type. The
method applies alpha spheres. This is a special
case of a contact sphere that circumscribes 4 atoms on its boundary and contain no internal atoms. Centers of alpha spheres are clustered into hydrophobic and hydrophilic areas.
The Alpha Sphere Method
MOE’s Site Finder employs an alpha shape construction algorithm whereby points on the protein’s surface are identified. The centres of spheres defined by combinations of four such points are marked. These clusters indicate potential binding sites on the protein’s surface, or voids within. Potential water sites are excluded. Sphere centroids are drawn in red if there is a probability for a hydrophilic contact on the surface of that sphere.
Identifies regions of tight atomic packing. This is not the same as locating pockets, since surface sites may still be regions of tight packing.
Filters out sites that are "too exposed" to solvent. In other words, sites that are on protrusions are unlikely to be good active sites.
Uses hydrophobic/hydrophilic classifications. This coarse classification of chemical type is used to separate water sites from the more likely hydrophobic sites.
Uses a definition of hydrophilic that is invariant to protonation state and tautomer state (this means no distinction between donor and acceptor atoms). Uses a definition of hydrophobic that is invariant to tautomer state (this means that aromaticity cannot be used).
Avoids grid-based methods since grid methods are not invariant to rotation of the atomic coordinates and can consume large amounts of memory.
Advantages:
- Uses no energy models
- Alpha spheres can be starting points for docking targets
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3. Binding Site Identification
MOE: Pharmacophore Derivation and Searching
Binding Site Identification
Geometric analysis of a molecule
Alpha spheres identify cavities
Sphere size is related to degree of exposure
Small spheres indicate "tight" cavities
The sphere size is not reflected in the graphical representation of centroids
Centroids are clustered for display
Hydrophilic contact points are marked
by red centroids
Hydrophobic (defined as
non-hydrophilic) are colored white.
P33 Protein Kinase
An alpha sphere describes the enclosed space between a set of four atoms. Thus the probability of exposure increases with the alpha sphere radius and small ones are more likely in regions of tight packing. Spheres with a radius of more than 5.0 Å are regarded as too exposed. The lower limit of the radius is specified by the "Probe Radius". The default settings of the probe and receptor contact radii for hydrophilic H-bond atoms (N,O) = 1.4 Å and for other hydrophobic atoms (C)=1.8. These contact radii are determined from examination of complexes in the PDB database. Adjust the radii in the settings panel.
The criteria if and how many spheres will be clustered are set by "Isolated Donor/Acceptor" and "Connection Distance". The first filter removes hydrophilic spheres if there is no hydrophobic sphere in the specified distance -> will be too hydrophilic. The second filter accumulates all alpha spheres within the connection distance. The actual default settings are heuristic and by no means “in stone”. Thus it may happen that two sites in the site list are considered as one single site. It is always advisable to inspect the full list of sites since it may be possible to extend individual sites with small molecule linkers into neighboring sites.
(see also panel description on next slides)
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4. MOE: Pharmacophore Derivation and Searching
Exercise: Query generation and Search from Receptor Site - Binding Site Identification I
Use alpha spheres to predict ligand positions
(MOE | File | Open) for the file 1ke6.moe
Open (MOE | Compute | Site Finder)
Click “Apply” on the Site Finder panel
17 sites are found …
Copyright © 2006 Chemical Computing Group Inc

5. MOE: Pharmacophore Derivation and Searching
Site Finder Panel
MOE: Pharmacophore Derivation and Searching
Site list:
Site: Site number
Size: Number of contributing spheres
Hyd: Number of hydrophobic atoms contacted in receptor
Side: Number of sidechain contact atoms
Residues: Residues at local surface
Atoms included in the calculation
Display mode of alpha spheres
Display mode of site(s); Residue selection option
Creates dummy atoms
Atoms: Specifies the atoms (receptor atoms, all atoms, selected/unselected atoms, selected residues/chains, currently loaded systems) which should be considered during site finding process. If Include Solvent is enabled, water molecules, salt ions, and common solvents will be included. If Visible Only is enabled then hidden atoms will be ignored. In all cases, atoms with the “Inert” attribute set will be ignored. Dummy atoms (LP atoms with no bonded neighbors) are automatically set inert.
Site List: When Apply is pressed, the calculated sites will be summarized in the list. Each site is numbered (starting from 1). The Residues column indicates the residues that make up the calculated site in the format chain:residue-name. The list is sorted by the Hyd column (descending order).
Render: The Render options control the display of the (selected) alpha spheres:
No Centers does not display the alpha spheres.
Alpha Centers draws a small sphere at each alpha sphere center.
Alpha Spheres draws a sphere of alpha radius at each center.
Red spheres indicate hydrophilic contacts, white spheres, hydrophobic. If Contact Atoms is on then all atoms within 4.5 Å of an alpha sphere will be selected.
Isolate: Controls how the atoms in the MOE window or the residues in the Sequence Editor will be treated when sites are selected:
None means that atoms and the backbone will be unaffected.
Atoms hides everything but the alpha spheres and atoms of the residues of the selected site(s).
Backbone displays the backbone cartoon of the residues of the selected site(s).
Atoms and Backbone hides everything but the alpha spheres and atoms of the residues of the selected site(s); site backbone is rendered as cartoon.
If SE Residues is on then the residues in the selected sites will be selected and all other residues deselected.
Dummies...: Pressing this button, a dummy atom will be created for each alpha sphere of the selected sites. The properties of the dummy atoms may be checked in the Atom Manager; The element type is LP and they are named either HYD (for hydrophobic) or LPA (for lone pair active; i.e., hydrophilic). In addition, the alpha sphere radius of the dummy atom is saved in the temperature factor (B). The spheres for a particular site are collected in a single residue that is named according to the site number (1 to x). All “residues” with the individual spheres are collected into a single chain.
Settings:
Probe Radius 1: The minimum radius (in angstroms) of a hypothetical hydrophilic hydrogen bonding atom (such as N or O).
Probe Radius 2: The minimum radius (in angstroms) of a hypothetical hydrophobic atom (such as C).
Isolated Donor/Acceptor: If there is no hydrophobic alpha sphere within the specified distance (in angstroms) to a hydrophilic alpha sphere then it is discarded. Set this value to a large number (e.g., 1000) if water sites are of interest.
Connection Distance: Individual alpha spheres are collected into separate sites by a single-linkage clustering algorithm. Two clusters are merged if there is a pair of spheres (one from each cluster) within the specified Connection Distance (in angstroms).
Minimum Site Size: After clustering, sites with fewer than the specified number of alpha spheres are discarded. Sites with a bounding sphere smaller than the Radius (in angstroms) are also discarded. Set both of these values to 0 if water sites are of interest.
Minimum sphere radii to detect (non-) LP-active atoms
Distance filter before clustering
Settings for alpha sphere clustering
Copyright © 2006 Chemical Computing Group Inc

6. MOE: Pharmacophore Derivation and Searching
Exercise: Query generation and Search from Receptor Site - Binding Site Identification II
4. Examine the different sites. The 2nd site has more receptor contacts but fewer hydrophobic contacts.
5. Select the first site. To restrict the view to the immediate environment by selecting Isolate: “Atoms” and enable SE Residues.
Ensure that (MOE | Selection | Synchronize) is ON. Invert the selection (MOE | Selection | Invert) and delete all hidden residues.
6. Keep the positions of the Alpha Centers by pressing “Dummies” and close the panel.
7. To increase the size of the dummy atoms <Ctrl>-click on one of the dummy atoms to select all of them. Select (Render | Space Filling).
Note to point 5:
a) If one wants to hide everything but the pocket (subsequent Ph4 derivation takes longer) select (MOE | Render | Hide | Selected)
b) If one wants to delete all atoms except the atoms of the pocket (Ph4 derivation is much faster) select (MOE | Edit | Delete)
Red: Potential hydrophilic contact areas
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7. MOE: Pharmacophore Derivation and Searching
Exercise: Query generation and Search from Receptor Site - Binding Site Identification III
8. Create a surface for the pocket using (MOE | Compute | Surfaces and Maps).
9. In the panel, keep default settings but select
Near: Dummy Atoms
Click: Apply
The colours of the surface displays those regions of the receptor surface suitable for a hydrogen bond or metal-lone-pair interactions.
Try also different surface color schemes to compare the results or modify the transparency (TF, TB) settings.
10. Save the pocket as 1ke6_pocket.moe
Copyright © 2006 Chemical Computing Group Inc

8. Receptor Contact Preferences
MOE: Pharmacophore Derivation and Searching
Receptor Contact Preferences
Objective
This approach complements the site finder information by identifying preferred areas for hydrophobic or hydrophilic interactions based on statistical preferences derived from non-bonding contacts in high resolution protein structures.
Methodology
Non-bonded protein-ligand interactions are analyzed with respect to distance, angle and out-of-plane preferences. The receptor and ligand atoms are classified into atom types and the experimental histograms of the contacts are fitted by analytical functions. Contour maps display likelihood ratios for hydrophobic over hydrophilic preferences (green) or vice versa (red).
Energy-based methods require knowledge of proton locations (tautomer and protonation state)
Advantages of contact preferences:
Transferable (meaningful) iso-contour levels
Values are probabilities in [0,1]
90% in one receptor is the same as 90% in another
Energy-based methods require adjustment of contour levels
Lone pair angle and out-of-plane angle supported
Directional preferences can be determined
Distance-only methodologies cannot capture angular preferences
De-localized pi systems are described very well
More robust statistics
Analytical forms require far less data to estimate parameters
Less frequent examples (e.g., halogens) modeled well
Heavy-atom only methodology
Partial charges and location of protons is not required
Copyright © 2006 Chemical Computing Group Inc

9. Receptor Atom Classification
MOE: Pharmacophore Derivation and Searching
Receptor Atom Classification
For each receptor atom, A, define a coordinate system
Define vectors u and v derived from hybridization and heavy neighbors
Some atom types do not have a u or a v (taken to be zero)
Define polar coordinate system from u and v
Distance from atom A r “distance”
Angle with u vector a “lone pair angle”
Angle with u in u-v plane p “out-of-plane angle”
A contact atom, B, is mapped to (r,a,p) local coordinates v u A v u
v taken from pi system A u A u v r a p A B
Copyright © 2006 Chemical Computing Group Inc

10. Receptor Atom Classification - Atom Typing
MOE: Pharmacophore Derivation and Searching
Receptor Atom Classification - Atom Typing
T_nQ2: HYD (50%) LPA (50%) r a
HYD LPA r lognorm 12-6 a gamma cauchy p gamma cauchy
The Fit Plots summarize the experimental (and fitted) data used to derive the contour levels. The experimental data were collected on high resolution pdb structures (better than 2 Å resolution) and includes sidechain-sidechain as well as sidechain-ligand contacts. Backbone atoms were excluded since they are conformationally too constrained.
Receptor atoms are classified according to element and chemical context (23 different types)
Receptor atom types depend on local coordinate system parameters
Avoids dependence on protonation state and tautomer state
Ligand atoms are classified into one of two types
LPA (“lone pair active”) for H-bond donors, acceptors and metals
HYD for all other atoms (hydrophobic or non-LPA)
Types allow to distinguish hydrophobic vs hydrophilic environments
The ligand atom types are invariant to protonation state and tautomer state. p
Copyright © 2006 Chemical Computing Group Inc

11. MOE: Pharmacophore Derivation and Searching
Exercise: Query generation and Search from Receptor Site - Contact Preferences
Contact preferences can be used to generate or refine a Ph4 query.
Hide the molecular surface from the previous exercise in (MOE | Window | Graphic Objects) to concentrate on contact potentials.
Select Surface: “Contact Preference” in the Surface and Maps panel and press Apply.
Play with different contour levels and display styles.
Contour levels are calculated as likelihood ratios. Decreasing down to 50 % there is no preference for hydrophobic over hydrophilic and vice versa. Increasing the level the more the maps will focus on those areas which are specific for hydrophobic or hydrophilic interactions. Those would be the most important matches to achieve high affinity. The figure displays exclusively the hydrophobic map at 87 %.
Compare the results (alpha spheres) of the Site Finder application with the calculated hydrophobic/hydrophilic contact potentials.
Add surface information (LP active color scheme) by showing hidden surfaces with the “Graphic Object Manager”.
Surface: The type of surface to generate: Gaussian Contact, Connolly (Analytic), Interaction (VDW), Contact Preference or Electrostatic Map. The particular selection affects the appearance of the bottom half of the Surfaces and Maps application.
Name: The name of the surface, which is created as a MOE Graphic Object.
Atoms: The atoms for which the particular surface or map is calculated; other atoms in the system will be ignored. By default, the Receptor Atoms are used (excluding ligands and solvent). If Visible Only is on, hidden atoms will be ignored. In all cases, dummy atoms and atoms flagged as inert will be ignored.
Near: Defines the atoms (Within a specified distance; by default 4.5 Angstroms) on which the generated graphics will be restricted, clipped, or truncated.
Press “?” to the right to briefly highlight the specified atoms in the MOE window.
Press the Isolate button to render the molecular system around the Near atoms and hide the remaining atoms of the system.
Level: Controls the appearance and iso-contour level of the Contact Preference maps. Each predictive interaction atom type (L_HYD for hydrophobic and L_HYD for hydrophilic) is controlled by:
Surface rendering mode: Line, Dot, Solid or None.
Color; select a color value to color the iso-contour surface of a particular interaction atom type.
An iso-contour level value (in percent). Use the slider or the text field to enter the display level. The generated surface will be all points in space for which the probability of finding an atom of a particular type versus the other types is equal to the specified value. The surface will dynamically update (in the MOE window). For example, if the L_HYD slider is set to 90%, the surface of points is displayed for which a contact atom will have a 90% probability of being hydrophobic.
Note that hydrogen atoms neither are required for Contact Preference Maps nor have an effect upon the calculation.
Copyright © 2006 Chemical Computing Group Inc

12. MOE: Pharmacophore Derivation and Searching
Exercise: Query generation and Search from Receptor Site - Pharmacophore Query Editor I
The suggestions for preferred interactions in the binding site may be used to derive a Ph4 query in absence of any known ligands.
Open (MOE | File | New | Pharmacophore Query), select an Ph4 scheme, e.g. PPCH_all.
Features are created in clusters of hydrophilic or hydrophobic groups.
Select individual dummy atoms to place the new feature. In the Query Editor, press Feature. A generic ‘Any’ feature will be created.
Adjust the feature positions by using <Shift><Alt><middle mouse button>.
Furthermore modify and reassign the feature types adjusting the radii, expressions, etc. according to chemical intuition.
Here, the reassignment of “any” features was performed by characterizing hydrophobic clusters to HydP and HydS and hydrophilic ones to DonP, DonS, AccP and AccS.
Copyright © 2006 Chemical Computing Group Inc

13. MOE: Pharmacophore Derivation and Searching
Exercise: Query generation and Search from Receptor Site - Pharmacophore Query Editor II
5. Once finished creating features, an excluded volume may be added.*) Use a surface representation to guide the adjustment of tolerances for the excluded volume.
*) Select all dummy atoms and invert the selection state (MOE popup | Invert). In the Ph4 Query Editor panel, select the Union button. Make sure the volume is set to Volume: Excluded.
This will draw an excluded volume spheres about the receptor pocket. Control (show, hide, toggle, etc.) the different graphical objects with (MOE | Window | Graphical Objects).
Copyright © 2006 Chemical Computing Group Inc

14. MOE: Pharmacophore Derivation and Searching
Exercise: Query generation and Search from Receptor Site - Pharmacophore Search
Save and use this query in the same way as the ones generated in the previous exercises.
Save the query as PPCH_ALL_sitefindV.ph4
Press Search and define the databases to be searched, consecutively double clicking on them, or select and then press Add.*)
Once the list is complete, press OK
The Search panel will reflect multiple databases. Continue with search process as previous exercises.
Note that the databases should be pre-annotated with the ph4 scheme
*) Here, more than one conformation databases (e.g. actives and inactives) are added to the search list.
Copyright © 2006 Chemical Computing Group Inc

15. Case Studies in Pharmacophore Search
MOE: Pharmacophore Derivation and Searching
Case Studies in Pharmacophore Search
Module 3: Structure-based design with known actives and structural binding site details
Case studies
Small molecules
Yes no
Protein structure No
Module 1
Module 3
Module 2
Module 1: Ligand based design with known actives in absence of structural binding site information
Module 2: Structure-based design with binding site information in absence of prior knowledge about active ligands
Module 3: Protein-ligand based design with known actives and structural binding site details
Copyright © 2006 Chemical Computing Group Inc

16. Module 3: Structure Based Ph4 Design
MOE: Pharmacophore Derivation and Searching
Module 3: Structure Based Ph4 Design
If structural information about both proteins and their ligands is available, the essential (conserved) protein ligand interactions (e.g. H-bonds) can be identified and used to focus on the key Ph4 features. Since those interactions include “projected” protein interaction sites, the results should be more meaningful than a small molecule alignment in itself.*)
MOE provides several applications to analyze protein-ligand information:
Alignment and superposition of proteins
Ph4 consensus analysis
Surface properties and contact preferences
In the Protein course:
Homology modeling (if only the amino acid sequence of a protein is available)
*) Note: Since all Ph4 relevant applications needed for this module are already discussed, it is compressed to the main workflow shown on the next slide (without giving exercises).
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17. Workflow of Structure Based Ph4 Design
MOE: Pharmacophore Derivation and Searching
Workflow of Structure Based Ph4 Design
Align a set of proteins with their co-crystallized ligands.
Ligands should already be docked in the binding site
Generate the relevant pharmacophore features based on SAR
Identify conserved Ph4 features with a Ph4 consensus analysis
Define the features based on biological/chemical knowledge of interactions
Identify key features based on interactions using Contact Statistics, Molecular Surfaces or Hydrogen Bonding interactions
Save pharmacophore model
Refine using Excluded volumes, or Interior Volumes for SMILES strings
Search conformational database(s) for ligands that contain the relevant pharmacophores
Re-design the pharmacophore model, if necessary and search again
Copyright © 2006 Chemical Computing Group Inc