Figure 2-1. Importing the input data for this tutorial.

1. Open Maestro and create a new project named macrocycle_tutorial.prj for this tutorial.
   • Don’t know how? See First steps in Maestro.
2. Download the tutorial zip file including input files and reference outputs here: https://www.schrodinger.com/sites/default/files/s3/release/current/Tutorials/zip/drug_development_macrocycles.zip
3. After downloading the zip file, unzip the contents in your Working Directorythe location where files are saved for ease of access throughout the tutorial.
4. Go to File > Import Structures.
5. Find and choose prepped_complex_2WER.maegz from the tutorial files.

Figure 3-1. Splitting the complex into Ligands, Water, Other.

1. In the Entriesa simplified view of the Project Table that allows you to perform basic operations such as selection and inclusion, right-click prepped_complex_2WER.
2. Choose Split > Into Ligands, Water, Other.
3. 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 prepped_complex_2WER_ligand1.

Figure 3-2. Setting up Prime Macrocycle Sampling calculation.

4. Type Z to fit the ligand to the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed.
5. Find and open Macrocycle Sampling panel from Tasks.
   • The Prime Macrocycle Sampling panel opens.
6. For Use structures from, choose Workspace.
7. For Sampling intensity (target # of conformers), choose Fast (100)
8. Change the Job name to macrocycle_2WER.
9. Click the Job Settings (cog) button.

Figure 3-3. Running the job.

10. For Incorporate, choose Append new entries as a new group.
11. For Total processors, type 4.
12. Click Run.
    • This job takes ~ 1 minute to run.
    • A banner appears when the job is incorporated.
    • You can find the results of this job in the tutorial files as macrocycle_2WER-out.maegz.
13. Close the Prime Macrocycle Sampling panel.

Figure 3-4. Opening the Superposition panel.

14. In the Entriesa simplified view of the Project Table that allows you to perform basic operations such as selection and inclusion, use ctrl+click (cmd+click) to also 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 group prepped_complex_2WER_ligand1 (Conformations) (39).
    • All entries in the group are 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.
    • prepped_complex_2WER_ligand1 remains 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.
15. Find and open Superposition panel from the Tasks.

Figure. 3-5. Superimposing selected entries.

16. Under Entries to superimpose, choose Project Table (selected).
17. For Reference structure, choose 2: prepped_complex_2WER_ligand1.
18. Check Add property to Project Table.
19. For Define structure for superposition using, choose ASL.
20. Click All.
21. Click Superimpose Structures.
    • RMS values are shown in the Results section.
    • Structures are superimposed.
22. Close the Superposition panel.

Figure 3-6. Including all entries from the group.

23. In the Entriesa simplified view of the Project Table that allows you to perform basic operations such as selection and inclusion, right-click on the group prepped_complex_2WER_ligand1 (Conformations) (39).
24. Choose Include.
25. In the Warning window, click Continue.
    • All entries in the group are includedthe entry is represented in the Workspace, the circle in the In column is blue in the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed.
    • Structures are somewhat aligned.

Figure 3-7. Choosing to show property in the Entries table.

26. In the Entriesa simplified view of the Project Table that allows you to perform basic operations such as selection and inclusion, click the three dots.
27. Choose Show Property.
    • The Show Property in Table dialog box opens.

Figure 3-8. Showing the Prime Energy property.

28. Click Choose.
29. In the search bar, type Prime.
30. Select Prime Energy and click OK.
    • The Prime Energy column is added to the Entriesa simplified view of the Project Table that allows you to perform basic operations such as selection and inclusion table.
31. Double-click the In circle of prepped_complex_2WER_protein
    • The protein is fixed in the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed.

 

Note: Click and drag the right edge of the Entriesa simplified view of the Project Table that allows you to perform basic operations such as selection and inclusion table to view these Prime Energies if necessary.

Figure 4-1. Opening the Ligand Docking panel.

1. Find and open Ligand Docking panel from Tasks.
   • The Ligand Docking panel opens.

 

Note: There are two ligands bound to the protein. In subsequent steps, either one of these ligand sites can be used for docking.

Figure 4-2. Loading the pre-generated receptor grid.

2. For Configure settings for, choose Macrocycles.
3. For Receptor grid, choose Browse file.
4. Navigate to and choose 2WER_glide-grid.zip from the tutorial files.
5. Click Open.

Figure 4-3. Loading the cognate ligand.

6. For Ligand source, choose File.
7. Click Choose.
8. Navigate to and choose randomized_radicicol.maegz from the tutorial files.
9. Click Open.

 

Note: The cognate ligand has been prepared using LigPrep and its initial conformation is essentially randomized so that it will not affect or bias the docking towards the cocrystal pose.

Figure 4-4. Viewing additional docking settings.

10. In the Docking options section, click Settings.
11. Confirm Sample macrocycles using Prime is enabled.

 

Note: This option requires a Prime license, but no macrocycle license is needed.

Figure 4-5. Write per-residue interaction scores and run.

12. In the Outputs section, click More Options.
13. Enable Write per-residue interaction scores.
14. Change the Job name to 2WER_glide-dock.
15. Click Run.
    • This job will take about 5 - 7 minutes to finish and automatically incorporate into the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed.
    • You can also find the results of this job in the tutorial files.
16. Close the Ligand Docking panel.

Figure 4-6. Visualizing the poses.

17. Optional: If you did not run the job yourself, you go to File > Import Structures and choose 2WER_glide-dock_pv.maegz from the tutorial files.
18. Click the Workflow icon next to 2WER_glide_dock_pv group header and choose View Poses.
    • The Pose Viewer panel opens.

Figure 4-7. The Pose Viewer panel.

19. For Type, choose Van der Waals.
    • The Van der Waals interactions between residues and the ligand are displayed.

 

Note: Residues are colored according to their interaction energies, ranging from green (favorable) to red (unfavorable). These energies are not quantitative and their meaningfulness is very limited due to the inherent limitations of rigid-receptor docking.

Figure 4-8. Applying ball-and-stick representation to ligand.

20. In Quick Select, click L.
    • The ligand is 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 Workspacethe 3D display area in the center of the main window, where molecular structures are displayed.
21. Go to the Style toolbox.
22. Choose ball-and-stick representation.
    • The ligand is rendered in ball-and-stick.

Figure 4-9. Good pose agreement between the cognate macrocycle (green) and the docked macrocycle (grey).

23. Ctrl+click (Cmd+click) to includethe entry is represented in the Workspace, the circle in the In column is blue prepped_complex_2WER_ligand1.
    • The crystal structure ligand is shown in the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed.
    • There is good pose agreement between the docked and cognate ligands.
24. Close the Pose Viewer panel.
25. Double-click the In circle of prepped_complex_2WER_protein.
    • The receptor is unfixed in the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed.

Figure 4-10. Superimpose without transformation.

26. Ctrl+click (Cmd+click) to includethe entry is represented in the Workspace, the circle in the In column is blue prepped_complex_2WER_ligand1 and randomized_radicicol in the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed.
27. Find and open Superposition panel from Tasks.
28. For Entries to superimpose, choose Workspace.
29. For Reference structure, choose prepped_complex_2WER_ligand1.
30. Check Compute without changing structures.

 

Note: Ensure Add Property to Project Table is still checked.

Figure 4-11. Get SMARTS from selection.

31. For Choose method, choose Substructures.
32. For Define structures for superposition using, choose SMARTS.
33. Double-click an atom of prepped_complex_2WER_ligand1
    • The whole cognate ligand is selected.
34. Click Get from Selection.
    • The SMARTS pattern of the ligand is generated.
35. Click Compute RMSD.
    • The RMSD between the docked and cognate poses is added to the bottom of the table in the Superposition panel (purple box in the figure).

 

The RMSD between the cognate and docked ligand is 0.55, indicating good agreement between the poses.

 

36. Close the Superposition panel.

Figure 5-1. Importing structures.

1. Click File > Import Structures.
2. Navigate to and choose over_combined.mae in the tutorial files.
3. Click Open.
   • Structures are added to the Entriesa simplified view of the Project Table that allows you to perform basic operations such as selection and inclusion.

Figure 5-2. Opening the Membrane Permeability panel.

4. Find and open Physics-Based Membrane Permeability panel from Tasks.
   • The Membrane Permeability Panel opens.

Figure 5-3. The Membrane Permeability panel.

5. Next to Use structures from, choose Project Table (200 selected entries).
6. Change Job name to membrane_permeability_over.
7. Optional: Click Run.
   • This job will take approximately 8 hours on 1 CPU.
   • You can find the results of this job in the tutorial files.

Figure 5-4. The Project Table.

8. Go to File > Import Structures.
9. Choose membrane_permeability_over-out.mae.
10. Click Open.
    • Structures are added to the Entriesa simplified view of the Project Table that allows you to perform basic operations such as selection and inclusion.
11. Click the Table button on the top right to open 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.

 

Note: 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 also be opened by pressing Ctrl+T/Cmd+T.

Figure 5-5. Showing the reference and predicted membrane permeability properties in the project table.

We have added an experimental reference value for the Caco2 membrane permeability (in cm/s x 10^-6), to which you can now compare the results of the calculation.

 

12. Hide all the properties.
13. Check the box next to Added by user.
    • The Caco2(cm/s) property is shown in 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.
14. Search for membrane.
15. Check the box to add Membrane dG Insert.
    • The Membrane dG Insert property is shown in 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.

Figure 5-6. The Project Table Calculator.

You need to convert the experimental reference to the same scale as the calculation by taking the logarithm.

 

16. In the top right corner of 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, open the Gadgets Menu and choose Equation Calculator.
    • The Property Calculator opens.
17. Fill in the name of the new property as log10_Caco2.
18. Click the Functions button and choose log10.
19. Click the Properties button and choose Caco2(cm/s).
20. Click ).

 

By default, the calculation would be applied to every Entry in 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, but you are only interested in the membrane_permeability_over-out group, so you can limit the calculation to the selected Entries.

 

21. Check the box for Selected Rows Only.
22. Click Execute.
    • A new column with the log of the Caco2 values is added to 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.

Figure 5-7. The Manage Plots panel.

Now, you can visualize the correlation between the prediction and the reference.

 

23. In the top right corner of the Project Table, open the Gadgets Menu and choose Chart Manager.
24. In the Manage Charts Panel, click Create and choose Scatterplot.

Figure 5-8. Preparing the scatter plot.

25. Choose log10 Caco2 for the x-axis.
26. Choose Membrane dG Insert for the y-axis.
27. Check Best fit line.
    • A best fit line is added to the scatter plot.
    • The equation of the Best fit line as well as the R² are now visible.
28. Check the box next to the Plot title.
    • The plot is now titled Membrane dG Insert vs. log10 Caco2.

 

Note: You can hover over the individual points in the scatter plot to see the structure of the corresponding ligand and identify and investigate outliers.

Figure 5-9. Saving the scatter plot.

Optional: You can now save your plot as a graphic to use outside of Maestro.

 

29. Change Save name to Membrane dG Insert vs. log10 Caco2.
30. Click Save.
    • A copy of the generated scatter plot is now saved in your desired destination.

Figure 6-1. Including 2e9p ligand, and 2e9p - minimized.

1. Go to File > Import Structures.
2. Choose 2E9P_cyclize.mae and click Open.
3. Use Shift+Click to include both 2e9p ligand and 2e9p - minimized.

 

Figure 6-2. Analyzing Workspace.

4. Find and open Ligand Designer panel from Tasks.
5. Click Analyze Workspace.
   • New entries are added to the Entriesa simplified view of the Project Table that allows you to perform basic operations such as selection and inclusion in the Ligand Designer group.
   • The Workspacethe 3D display area in the center of the main window, where molecular structures are displayed now has just a single ligand and a cloud surrounding it to represent the growth space.

Note: You can uncheck Adjust view when analyzing to retain the previous view in the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed.

Figure 6-3. Selecting Cyclize Ligands.

6. Click Workflows and select Macrocyclization.
7. Click Enumerate.
   • The exact number of returned ligands and the duration of the job can vary depending on the software version you are using.
   • You can find a subset of the results of the macrocyclization in the outputs folder of the tutorial files as 2e9p_cyclized_ligands.maegz.

Note: The darker blue growth space indicates the region is solvent exposed, and the lighter blow indicates more buried regions in the protein.

Figure 6-4. Scrolling through the enumerated macrocycles.

8. Click the right arrow in the Ligand Designer.
   • C_1 is now included in the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed.
   • The radar plot updates with the properties of the new compound.

Note: If you want to see the cyclized ligands in the context of the binding pocket, you can toggle on the Ligand-Receptor interactions or include the 2e9p - minimized protein structure in the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed.

Figure 6-5. Tiled view of the Ligand Designer outputs.

See the Forming Protein-Ligand Interactions with the Ligand Designer tutorial for examples of how to sort and filter enumeration outputs.

 

9. Use Shift+Click to include all of the macrocycles.
   • The enumerated macrocycles are all overlaid in the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed.
10. Press Ctrl+L (Cmd+L) to tile the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed.
    • The enumerated structures are visualized.
11. Press Ctrl+L (Cmd+L) to remove the tiles in the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed.
12. Close the Ligand Designer panel.

Figure 7-1. Selecting atoms important for activity.

1. 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 Cyclized - 2e9p ligand 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 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 2e9p ligand.
3. Exclude any other entries from the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed.

 

Note: If you did not run the enumeration in the last section yourself, you can find a subset of the enumerated ligands in the outputs directory of the tutorial files as 2e9p_cyclized_ligands.maegz and import them.

 

Figure 7-2. Selecting the un-cyclized ligand atoms.

4. In the Workspace, select all the atoms from 2e9p ligand from O₈ to O₂₀.

Figure 7-3. Opening the Macrocycle Propensity option in Structure Analysis.

5. Find and open Macrocycle Propensity from Tasks.
   • The Macrocycle Bioactive Conformation Propensity panel opens.

Figure 7-4. The Macrocycle Bioactive Conformation Propensity panel.

6. For Use macrocycles from, choose Project Table.
7. For Sampling, select Perform macrocycle sampling.
8. For Use reference from, choose Workspace (included entry).

 

Figure 7-5. Running the macrocycle bioactive conformation propensity job.

9. For Common atoms important for activity, select Get from Workspace Selection.
10. Change the Job name to macro_2e9p_propensity.
11. Optional: Click Run.
    1. This job takes ~ 2 hours to finish with 2 processors.
    2. A new group is added to the Entriesa simplified view of the Project Table that allows you to perform basic operations such as selection and inclusion.
    3. You can find the results of this job in the outputs directory of the tutorial files.

Figure 7-6. Opening the Project Table.

 

If you did not run the job yourself, you can find the results for a subset of the cyclized ligands in the tutorial files.

 

12. Go to File > Import Structures
13. Find and choose macro_2e9p_propensity_sampling-out.maegz from the tutorial files.
14. In Maestro, click Table.
    1. 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 panel opens.

Figure 7-7. Adding properties to the Project Table through the Property Tree.

15. In the Property Tree, search for stability, and select stability prob metric below thresh and stability metric expval.
    1. Stability prob metric below thresh and stability metric expval are added to 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.

Figure 7-8. Viewing properties in the Project Table.

Determining whether “stability metric expval” or “stability prob metric below thresh” would be most useful in assessing the viability of various linkers depends largely on the specifics of the system. For systems where low RMSD is associated with better binding, expval is a more useful value to follow. For systems where the more often you find low RMSD is associated with better binding, stability prob metric below thresh is a more useful value to follow. Usually these values agree with each other, but when they do not, it is best to work off of prior knowledge of the system.

Figure 8-1. Import structures for FEP+.

1. Go to File > Import Structures and choose Macrocycle_Ligands.mae from the tutorial files.

Figure 8-2. Opening the FEP+ panel.

2. Find and open FEP+ panel from Tasks.

Figure 8-3. Importing structures.

3. For Import structures or perturbation map from, choose Project Table (4 selected entries).
4. Click Import.
   • The Receptor name and Total ligands populates.

 

If the automatic checks identify potential issues with the receptor or missing torsion parameters for the ligands, you would see yellow warning triangles rather than the green OK and checkmark. You could then investigate and potentially correct the issues by hovering over or clicking the warning signs.

 

5. Click Relative.

Figure 8-4. Adding the affinity data.

6. Switch to the Map tab.
7. Click Affinity and choose Experimental Data.
   • The Choose Affinity Property dialog box opens.
8. Choose exp dG (user).
9. Click OK.
   • Experimental affinity data is added to the Summary table.
10. Click Generate Map
    • The Map Options panel opens.

Figure 8-5. Generating Map.

11. Next to Map topology type, choose Optimal.
12. Click Generate Map.
    • This job will take ~ 1 minute to run.
    • A map is generated connecting the ligands.

Figure 8-6. Displaying perturbation properties and changing the Job name.

13. Click Display perturbation properties.
14. Choose Experimental ddG and Similarity scores.
    • The values are shown on the map edges.
15. Change the Job name to fep_mapper_5ANT.

Figure 8-7. FEP+ Advanced Options dialog box.

A standard 5 ns production run can frequently be too short for macrocycles, so we recommend running all legs for 20 ns.

 

16. Click Settings.
17. In the Simulation Parameters section, change the Simulation time for the Complex stage to 20.0.
18. Check the box for match to apply the same simulation length to all stages.

 

Note: If your ligands are missing torsion parameters, you can check the Generate missing parameters with Force Field Builder option to run parametrization as part of the FEP+ job. If you already have parameters for your ligands, you can check the Use customized version option and load them in by clicking the three dot button.

 

19. Click OK.

Figure 8-8. Changing the job settings.

This is a compute-intensive job, so you may want to run it on a high-performance computing cluster. You can specify the CPU and GPU hosts to use here.

 

20. Click the Job Settings (cog) icon.
    • The Job Settings dialog box opens.
21. Choose your CPU host and GPU host.
22. Do not click Run.
    • To save time, you will look at the pre-generated results.

Figure 8-9. Opening the FEP+ output.

23. Find and open FEP+ panel from Tasks.
24. For Import structures or perturbation map from, choose File.
25. Click Browse.
26. Choose fep_mapper_5ANT_out.fmp.
27. Click Next.

 

Figure 8-10. Viewing the Correlation Plot.

A first look should go to the correlation plot, although with only three ligands in the map, the associated statistics are not robust.

 

28. Click Plot.
    • The Correlation Plot (FEP+) panel opens.

Figure 8-11. Viewing details for each edge in the map.

You can check the Analysis tab for a more detailed overview of the individual edges in the map and identifying potential issues.

 

29. In the FEP+ panel, go to the Analysis tab.
30. Hover your mouse pointer over one of the cells labeled Bad to see more details.
31. Optional: Click the View button in the Edge Analysis column for one of the edges to dig deeper into potential issues.

 

For more information on FEP+ results analysis see the BACE1 Inhibitor Design Using Free Energy Perturbation tutorial.