Identifying Binding Site Requirements and Lead Optimization with WaterMap

Tutorial Created with Software Release: 2023-4
Topics: Hit-to-Lead & Lead Optimization, Small Molecule Drug Discovery, Structure Prediction & Target Enablement
Products Used: WaterMap

Tutorial files

40 MB
This tutorial is written for use with a 3-button mouse with a scroll wheel.
Words found in the Glossary of Terms are shown like this: Workspacethe 3D display area in the center of the main window, where molecular structures are displayedthe 3D display area in the center of the main window, where molecular structures are displayed

 

Tip: You can hover over a glossary term to display its definition. You can click on an image to expand it in the page.
Abstract:

 

In this tutorial, you will learn how to perform and analyze the results from WaterMap Analysis and WM/MM Scoring within the Maestro interface. A retrospective “lead-optimization” strategy is performed, where known binders are overlaid with the results from WaterMap. Sites are identified for water replacement, water displacement, or water coordination. The results from visual inspection are further confirmed by WaterMap/Molecular Mechanics Scoring, whereby the active site residues around the bound ligands are allowed to relax and breathe to better accommodate the congeneric series of ligands.

 

Tutorial Content

  1. Creating Projects and Importing Structures

  1. Understanding Binding Site Requirements for WaterMap

  1. Retrospective “Lead Optimization” with WaterMap

  1. Conclusion and References

  1. Glossary of Terms

1. Creating Projects and Importing Structures

At the start of the session, change the file path to your chosen Working Directorythe location that files are saved in Maestro to make file navigation easier. Each session in Maestro begins with a default Scratch Projecta temporary project in which work is not saved, closing a scratch project removes all current work and begins a new scratch project, which is not saved. A Maestro project stores all your data and has a .prj extension. A project may contain numerous entries corresponding to imported structures, as well as the output of modeling-related tasks. Once a project is created, the project is automatically saved each time a change is made.

Structures can be imported from the PDB directly, or from your Working Directorythe location that files are saved using File > Import Structures, and are added to the Entriesa simplified view of the Project Table that allows you to perform basic operations such as selection and inclusion and Project Tabledisplays the contents of a project and is also an interface for performing operations on selected entries, viewing properties, and organizing structures and data. The Entriesa simplified view of the Project Table that allows you to perform basic operations such as selection and inclusion is located to the left of the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed. The Project Tabledisplays the contents of a project and is also an interface for performing operations on selected entries, viewing properties, and organizing structures and data can be accessed by Ctrl+T (Cmd+T) or Window > Project Table if you would like to see an expanded view of your project data.

  1. Double-click the Maestro icon.

Figure 1-1. Change Working Directory option.

  1. Go to File > Change Working Directory.
  2. Find your directory, and click Choose.
  3. Pre-generated input and results files are included for running jobs or examining output. Download the zip file here: https://www.schrodinger.com/sites/default/files/s3/release/current/Tutorials/zip/watermap_bindingsite_leadopt.zip
  4. After downloading the zip file, unzip the contents in your Working Directorythe location that files are saved for ease of access throughout the tutorial.

Figure 1-2. The Open Project panel.

  1. Go to  File > Open Project.
  2. Select Lead_Opt_WM.prjzip.
  3. Click Open.
  4. In the Save scratch project dialog box, click OK.

Note: Banners appear when files have been imported, jobs incorporated into the Entriesa simplified view of the Project Table that allows you to perform basic operations such as selection and inclusion, or to prompt a common next step.

Note: By default the structure corresponding to the imported file is both includedthe entry is represented in the Workspace, the circle in the In column is blue in the Workspace the 3D display area in the center of the main window, where molecular structures are displayedand selected(1) the atoms are chosen in the Workspace. These atoms are referred to as "the selection" or "the atom selection". Workspace operations are performed on the selected atoms. (2) The entry is chosen in the Entries (and Project Table) and the row for the entry is highlighted. Project operations are performed on all selected entries in the Entriesa simplified view of the Project Table that allows you to perform basic operations such as selection and inclusion. Please refer to the Glossary of Terms for the difference between includedthe entry is represented in the Workspace, the circle in the In column is blue and selected(1) the atoms are chosen in the Workspace. These atoms are referred to as "the selection" or "the atom selection". Workspace operations are performed on the selected atoms. (2) The entry is chosen in the Entries (and Project Table) and the row for the entry is highlighted. Project operations are performed on all selected entries.

Figure 1-3. Saving the project in the Save Project panel.

  1. Go to File > Save Project As.
  2. Change the File name to Binding_Site_WM_tutorial.
  3. Click Save.
    • The project is now named Binding_Site_WM_tutorial.prj

2. Understanding Binding Site Requirements with WaterMap

WaterMap can be used to assess the druggability of a binding site by determining its hydration-site thermodynamics. For more information on WaterMap, see the WaterMap User Manual. The preparation of a system prior to performing a WaterMap calculation is extremely important and can have a profound influence on the quality of results that emerge from it. In this example, all the crystallographic waters were maintained for WaterMap analysis. For more information on preparing a protein with Protein Preparation Workflow, see the  Introduction to Structure Visualization and Preparation tutorial. Any unusual residue charges or incorrectly flipped residues can drastically alter the hydration structure seen in the final WaterMap. WaterMaps can help us determine if it makes sense to displace, replace, or interact with a water molecule in a high energy hydration-site. Please see the Target Analysis with SiteMap and WaterMap Tutorial for guidance on when each is appropriate.

2.1 Set up and run WaterMap jobs

Figure 2-1. Include the pre-prepped 3RLP in the Workspace

  1. Expand 3RLP and 3RLP_prepared-out1 groups in the Entriesa simplified view of the Project Table that allows you to perform basic operations such as selection and inclusion.
  2. Includethe entry is represented in the Workspace, the circle in the In column is blue and select(1) the atoms are chosen in the Workspace. These atoms are referred to as "the selection" or "the atom selection". Workspace operations are performed on the selected atoms. (2) The entry is chosen in the Entries (and Project Table) and the row for the entry is highlighted. Project operations are performed on all selected entries 3RLP - prepared.
    • If you do not see the structure in the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed, click the Fit-all button.

 

Note: The 3RLP – prepared entry has been preprocessed, optimized, and minimized using the Protein Preparation Workflow. Chain A was chosen from the PDB and all the water molecules were retained.

 

  1. Go to Tasks > Browse > WaterMap > Perform Calculations.
    • The WaterMap - Perform Calculation panel opens.

Figure 2-2. Setting up a WaterMap job.

  1. Under Binding site definition, choose Ligand and check the box for Pick.
    • A banner prompts you to pick an atom.
  2. Click on any ligand atom in the 3D Workspacethe 3D display area in the center of the main window, where molecular structures are displayed.
    • The ligand is selected.

 

Truncation can lead to misleading results if it goes through a critical part of the protein. We recommend considering truncation only if working with a very large system, calculation time is a concern, and allostery is not thought to play a significant role in the protein.

 

  1. Uncheck Truncate protein.
  1. Change Job name to watermap_3RLP.
  2. Click Run.
    • This job will take ~ 2 hours.
    • To save time, you will look at the pre-generated results from a previous release. Your results may vary slightly from the ones shown.

2.2 Analyze water structures

The thermodynamic profile for each of the hydration sites can be used in ligand design. In this section, results from a WaterMap calculation performed on the HSP-90 dataset are analyzed. This section aims to demonstrate how the explicit use of WaterMap could have been used to design these HSP-90 targeting ligands.

Figure 2-3. Including the entries for analyzing the result.

  1. Expand watermap_3RLP_wm group in the Entriesa simplified view of the Project Table that allows you to perform basic operations such as selection and inclusion.
  2. Includethe entry is represented in the Workspace, the circle in the In column is blue and fix watermap_3RLP, sampling_coordinates, and 3RLP - prepared by double clicking on the corresponding In column.
    • A pin appears alongside the corresponding entries, which indicates entries are fixed in the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed.
    • watermap_3RLP contains dummy atoms where the oxygens of the waters are located within the active site from molecular dynamics simulations. This is the WaterMap output.
    • Sampling_coordinates contains the ligand from the original 3RLP PDB structure
    • 3RLP – prepared contains the protein structure from the prepared, optimized and minimized 3RLP structure.

 

Note: By default the WaterMap entry includes points where the oxygen of the water exists at the average structure. The default representation is a wire representation and the labels for the energies of each water are also displayed.

Figure 2-4. Expanding and selecting the HSP-90 LIGANDS group.

  1. In the Entriesa simplified view of the Project Table that allows you to perform basic operations such as selection and inclusion, expand and select(1) the atoms are chosen in the Workspace. These atoms are referred to as "the selection" or "the atom selection". Workspace operations are performed on the selected atoms. (2) The entry is chosen in the Entries (and Project Table) and the row for the entry is highlighted. Project operations are performed on all selected entries the HSP-90 LIGANDS group.
  • This group contains all the  inhibitors (from Kung et al.) reasonably modeled into HSP-90.
  • All the ligands are denoted by numbers, sequentially from 1-9 to align with ligands 9-17 as per the notation in the publication.

Figure 2-5. The WaterMap - Examine Results Panel to label the sites based on their free energies and view the WaterMap results.

  1. Go to Tasks > Browse > WaterMap > Examine Results.
    • The WaterMap - Examine Results panel opens.
  2. Click on Analyze Workspace button to populate the panel with the WaterMap data.
  3. For Color sites by, choose ∆G (simple).
  4. Check Show color in table.
    • The color associated with each ∆G for each water site will appear in the ∆G column.

In the WaterMap - Examine Results panel, the top half of the panel (including the table and above) changes how the WaterMap is displayed in the Maestro Workspace. The bottom half of the panel has options to allow for changes in which water sites are displayed and to score ligands based on WaterMap results. See WaterMap - Examine Results Panel for more information.

Figure 2-6. WaterMap sites colored by absolute free energies and labeled by site number.

  1. For Site label, choose Site number.
    • Each water now has a corresponding number in the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed.

 

Note: The representation is colored from red being highest in free energy and green lowest in free energy. The majority of the displaceable waters are surrounding the pyrimidine moiety.

 

 

3. Retrospective “Lead Optimization” with WaterMap

3.1 Analyze Water Energetics in Ligand Design

In this section, we use WaterMap to retrospectively guide the design of the ligands. We will start with compound 1 (R4 and R5 are hydrogens) and bring various ligands into the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed along with the WaterMap to examine how they displace/replace various water molecules. The basic 2D structure of the ligands derived from compound 1 is shown to the right. R4 and R5 are the functional groups that are added to the pyrrolopyrimidine. We will see how the information from WaterMap can be used to explain the functional group substitutions by employing a displacement or replacement strategy.

Figure 3-1. Focusing on the WaterMap sites with the most negative ΔG.

  1. In the WaterMap - Examine Results panel, click on the ΔG column to organize the table from highest to lowest ΔG.
  2. Select the highest ΔG in the table (most positive) and shift-click to site 29 at ΔG ~ -0.67.
  3. Select Show only Selected Rows.

Figure 3-2. Select only the WaterMap sites with ΔG greater than 2.0 kcal/mol.

  1. In the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed, inspect the locations of the waters with the most positive free energies.
    • If you choose the site you would like to investigate in the Examine Results panel, you can then zoom to selection using the Quick Select tools. Just remember to deselect Show Only Selected Rows after.

There are two red colored sites very close to the ligand, site 3 with ΔG ~5.43 kcal/mol and site 8 with a ΔG ~5.13. These hydration sites can be exploited for a classic case of water displacement in a hydrophobic region. Sterically complementary groups (R4 and R5) can be added to the ligand in these positions.

Figure 3-3. Displaying the ligand in thick tube representation.

  1. Under Quick Select, click L.
    • All the atoms of ligand are selected.
  2. Go to the Style Toolbox and choose thick tube representation.
  3. Identify the larger spheres which represent the WaterMap sites 3 and 8
    • The crystallized ligand already overlaps with sites 44 and 54 in the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed.

Optional: With the original ligand included and fixed in (pinned) the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed, include the entry is represented in the Workspace, the circle in the In column is blueCompound 1 in the HSP-90 Ligands group in the Workspace. See Table 1 for Compound Progression, or the tuning of compounds from hits to leads, with WaterMap insight.

Figure 3-4. The WaterMap sites 3 and 8, and distances to nearest atoms of 4-(2,4-dichloro-5-methoxyphenyl)-6-methylpyrimidin-2-amine.

  1. Select Measure from the Favorites Toolbar.

Note: You can also go to Tasks and search for Measure, which will be a Workspace Operation.

  1. Click on the primary amine and WaterMap site 3 in the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed.
  2. Perform distance measurements between the secondary amine and WaterMap site 8.
  3. Click OK.
    • The Measure dialog closes.

Measuring distances can give information on types of substituents for replacing or displacing the waters in the active site. Visual inspection can be compared with the following table to generate a hypothesis of compound progression.

 

 

WaterMap only computes the energetics associated with waters around the binding site, while the experimental binding energy includes all components of binding, so a direct correlation may not be observed if water thermodynamics are not the dominant factor for a particular ligand.

3.2 Scoring Ligand Poses with WM/MM

The WM/MM scoring approach uses a combination of WaterMap and MM-GBSA to give a reasonably thorough model of the protein-ligand binding process. It might be difficult to determine the optimum pose through docking. In such cases, it makes sense to carry multiple representative ligand poses into the WM/MM calculation and use the best scoring pose for the final assessment.

Figure 3-5. The WM/MM Scoring panel for refining the ligand/protein binding site structure for WM/MM scoring.

  1. Go to the WaterMap - Examine Results panel.
  2. Click Perform WM/MM Scoring at the bottom of the panel.
    • The WM/MM Scoring panel opens.
  3. Select entries for ligands from the selected(1) the atoms are chosen in the Workspace. These atoms are referred to as "the selection" or "the atom selection". Workspace operations are performed on the selected atoms. (2) The entry is chosen in the Entries (and Project Table) and the row for the entry is highlighted. Project operations are performed on all selected entries group HSP90 Ligands from the Project Table.
  4. Take the WaterMap structures from the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed entry.
  5. Allow for protein flexibility within 7.0 Å from the ligand during WM/MM scoring.
  6. Change the Job name to 3RLP_WM_MM_Scoring.
  7. Click Run.
    • This calculation takes a few minutes.
    • To save time, you will look at the pre-generated results.
  8. Close the WaterMap - Examine Results panel.

Figure 3-6. Comparing Compound 1 before (orange) and after (green) WM/MM structure optimization.

  1. Unpin 3RLP - prepared and sampling_coordinates from the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed.
  2. Select(1) the atoms are chosen in the Workspace. These atoms are referred to as "the selection" or "the atom selection". Workspace operations are performed on the selected atoms. (2) The entry is chosen in the Entries (and Project Table) and the row for the entry is highlighted. Project operations are performed on all selected entries the 3RLP_WM_MM_Scoring group in the Entriesa simplified view of the Project Table that allows you to perform basic operations such as selection and inclusion.
  3. Includethe entry is represented in the Workspace, the circle in the In column is blue Compound 1 from group HSP-90 Ligands and 3RLP_WM_MM_Scoring.
    • Compare the slight structural differences witnessed in the ligand binding site upon structure optimization with WM/MM.
  4. Include Compound 1 from the 3RLP_WM_MM_Scoring-out group and display it in the thick tube representation.

Figure 3-7. The Show Property option.

 

  1. In the Entriesa simplified view of the Project Table that allows you to perform basic operations such as selection and inclusion, click on the Change table settings (three vertical dots) and choose Show Property.
    • The Show Properties in Table dialog box opens.

Figure 3-8. The predicted deltaG of each ligand binding from WaterMap alone versus WM/MM2.1.

  1. Search and add the following Properties to the Entriesa simplified view of the Project Table that allows you to perform basic operations such as selection and inclusion table:
    • WM ligand -T*deltaS
    • WM ligand deltaG
    • WM/MM2.1 dG Bind
  2. Compare the values in the WM/MM2.1 dG Bind to WM ligand deltaG.
    • The WaterMap ligand deltaG is significantly underpredicting the deltaG of binding compared to WM/MM2.1 dG.

Small conformational rearrangements and breathing of the protein residues around each ligand can contribute significantly to the stability of a ligand in the binding pocket. The values from WM/MM2.1 reflect similar variations across compounds as was seen with the experimental Ki values (Table 1).

4. Conclusion and References

In this tutorial, you performed and analyzed the results from WaterMap Analysis and WM/MM Scoring in the Maestro interface. A retrospective “lead-optimization” strategy was performed, where known binders were overlaid with the results from WaterMap. Sites were identified for water replacement, water displacement, or water coordination. The results from visual inspection were further confirmed by WaterMap/Molecular Mechanics Scoring, whereby the active site residues around the bound ligands were allowed to relax and breathe to better accommodate the congeneric series of ligands.

For further reading:

 

 

5. Glossary of Terms

Entries - a simplified view of the Project Table that allows you to perform basic operations such as selection and inclusion

included - the entry is represented in the Workspace, the circle in the In column is blue

Project Table - displays the contents of a project and is also an interface for performing operations on selected entries, viewing properties, and organizing structures and data

Scratch Project - a temporary project in which work is not saved, closing a scratch project removes all current work and begins a new scratch project

selected - (1) the atoms are chosen in the Workspace. These atoms are referred to as "the selection" or "the atom selection". Workspace operations are performed on the selected atoms. (2) The entry is chosen in the Entries (and Project Table) and the row for the entry is highlighted. Project operations are performed on all selected entries

Working Directory - the location that files are saved

Workspace - the 3D display area in the center of the main window, where molecular structures are displayed