Drug Development with Macrocycles

1. Double-click the Maestro icon.
   • (No icon? See Starting Maestro)

Figure 2-1. Change Working Directory.

2. Go to File > Change Working Directory.
3. Find your directory, and click Choose.
4. 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/drug_development_macrocycles.zip
5. After downloading the zip file, unzip the contents in your Working Directorythe location where files are saved for ease of access throughout the tutorial.

Figure 2-2. Saving the Project.

6. Go to File > Save Project As.
7. Change the File name to macrocycle_tutorial.
8. Click Save.
   • The project is named macrocycle_tutorial.prj.

Figure 2-3. Importing the prepared structure.

9. Go to File > Import Structures.
10. Choose prepped_complex_2WER.
11. Click Open.
    • Structure is in the Entriesa simplified view of the Project Table that allows you to perform basic operations such as selection and inclusion and Workspace.

Note: To save time, this structure has already been prepared using the Protein Preparation Workflow.

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.
   • Four new entries are added.

Figure 3-2. Opening the Macrocycle Sampling panel.

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.
4. Type Z to fit the ligand to the Workspace.
5. Find and open the Macrocycle Sampling panel from the Tasks.

Figure 3-3. Set up Prime Macrocycle Sampling calculation.

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 3-4. Modifying the job settings and running the job.

10. For Incorporate, choose Append new entries as a new group.
11. For Total, type 4.
12. Click Run.
    • This job takes ~ 1 minute to run
    • A banner appears when the job is incorporated.
    • A new group prepped_complex_2WER_ligand1 (Conformations) is added to the Entriesa simplified view of the Project Table that allows you to perform basic operations such as selection and inclusion.
13. Close the Prime Macrocycle Sampling panel.

Figure 3-5. Opening the Superposition panel.

14. Click View in Project in the banner.
    • All the entries in the newly added group are selected in the Entriesa simplified view of the Project Table that allows you to perform basic operations such as selection and inclusion table.
15. 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 prepped_complex_2WER_ligand1.
16. Find and open the Superposition panel from the Tasks.

Figure. 3-6. Superimposing the selected entries.

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

Figure 3-7. Including all entries from the group.

24. In the Entriesa simplified view of the Project Table that allows you to perform basic operations such as selection and inclusion, right-click the group prepped_complex_2WER_ligand1 (Conformations) (39).
25. Choose Include.
26. In the Warning box, 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 Workspace.
    • Structures are somewhat aligned.

Figure 3-8. Choosing to Show Property in the Entries table.

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

Figure 3-9. Adding Prime Energy property to the Entries table.

29. Click Choose.
30. In the search bar, type Prime.
31. Choose Prime Energy and click OK.
    • Prime Energy is added to the Entriesa simplified view of the Project Table that allows you to perform basic operations such as selection and inclusion table.
    • You may need to click and drag the edge of the Entriesa simplified view of the Project Table that allows you to perform basic operations such as selection and inclusion table toward the right to see the added property.
32. Double-click the In circle of prepped_complex_2WER_protein
    • The protein is fixed in the Workspace.

Figure 4-1. Opening the Macrocycle Docking panel.

1. Includethe entry is represented in the Workspace, the circle in the In column is blue prepped_complex_2WER in the Workspace.
2. Find and open the Macrocycle Docking panel from the Tasks.

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 receptor and the ligand.

3. For Receptor, click Load Workspace.
4. For Box center, choose Centroid of ligand.

Note: By default, the panel selects a ligand to center the box around. To specify a different ligand when multiple ligands are present, check the "Pick" box and select your preferred ligand from the Workspace.

5. For Ligands to dock, choose File and click Browse next to it.
6. Navigate to and choose randomized_radicicol.maegz in your Working Directorythe location where files are saved.

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 4-3. Modifying the Job name and running the job.

7. Change the Job name to macrocycle_docking_2WER-cognate.
8. Click Run.
   • This job takes ~ 10 minutes to finish.
   • Pre-generated results are included in the tutorial files.
   • A banner appears once the job is completed and a new group macrocycle_docking_2WER-cognate-out is added to the Entriesa simplified view of the Project Table that allows you to perform basic operations such as selection and inclusion table.
9. Close the Macrocycle Docking panel.

Figure 4-4. Setting up the Workspace and applying Binding Mode Comparison Preset to visualize the docked poses.

Optional: If you did not run the job yourself, you go to File > Import Structures and choose macrocycle_docking_2WER-cognate-out.maegz from the tutorial files.

10. Double-click the In circle of the prepped_complex_2WER to fix it.
11. Shift+click to includethe entry is represented in the Workspace, the circle in the In column is blue all the docked poses in the Workspace.
12. Go to Presets and choose Binding Mode Comparison.
    • Take your time to step through the docked poses.

Figure 5-1. Selecting structures in the Entries table.

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 the Membrane Permeability panel from the Tasks.

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 is time intensive.
   • The pregenerated results are included in the tutorial files.
   • To save time, you will look at the pregenerated results.

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. Click the All checkbox twice to hide all the properties.
13. Expand All and further expand Added by user and check the box next to it.
    • 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.

Figure 5-6. Adding Membrane dG Insert property to the table.

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 5-7. 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 5-8. 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 5-9. 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 5-10. 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. Navigate to and choose 2E9P_cyclize.mae and click Open.
3. Use Shift+Click to include both 2e9p ligand and 2e9p - minimized.

Figure 6-2. Analyzing the Workspace.

4. Find and open the Ligand Designer panel from the Tasks.
5. Click Analyze Workspace.
   • A new group Ligand Designer  with two entries is added to the Entriesa simplified view of the Project Table that allows you to perform basic operations such as selection and inclusion table.
   • The Workspace 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 Workspace.

Figure 6-3. Select 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 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 blue 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 Workspace.
   • 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 Workspace.

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

9. Right-click the Cyclized – 2e9p ligand group and choose Include to includethe entry is represented in the Workspace, the circle in the In column is blue all the entries in the Workspace.
   • The enumerated macrocycles are all overlaid in the Workspace.
10. Type Ctrl+L (Cmd+L) to tile the Workspace.
    • The enumerated structures are visualized.
11. Close the Ligand Designer panel.
12. Right-click anywhere in the empty workspace and choose Clear Workspace.

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

Figure 7-1. Setting up the Workspace.

1. Includethe entry is represented in the Workspace, the circle in the In column is blue 2e9p ligand in the Workspace.
2. Ctrl+click (Cmd+click) to select 2e9p ligand in the Entriesa simplified view of the Project Table that allows you to perform basic operations such as selection and inclusion.

Note: If you did not run the enumeration in the last section yourself, you can find 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.

3. In the Workspace, select all the atoms from 2e9p ligand from 0₈ to 0₂₀.

Figure 7-3. Opening the Macrocycle Bioactive Conformation Propensity panel.

5. Find and open the Macrocycle Propensity panel from the Tasks.
   1. 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.
    • This job is time intensive. We advise against running this job.
    • The pregenerated results are included in the tutorial files.
    • To save time, you will look at the pregenerated results.

Figure 7-6. Opening the Project Table.

12. Go to File > Import Structures.
13. Navigate to and choose macro_2e9p_propensity_stability-out.maegz and click Open.
    • This may take some time.
14. Open the Project Table.

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

15. In the Property Tree, search for “stability”, and check the boxes for stability prob metric below thresh and stability metric expval.
    • 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.
16. Close 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 once you have finished viewing the properties.

Figure 8-1. Importing structures for FEP+.

1. Click File > Import Structures.
2. Navigate to and choose Macrocycle_Ligands.mae in the tutorial files.
3. Click Open.

Figure 8-2. Opening the FEP+ panel.

4. Find and open the FEP+ panel from the Tasks.

Figure 8-3. Importing structures from the Project Table.

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

Note: 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. If needed, run Force Field Builder if needed before proceeding with the calculation.

7. Click Relative.

Figure 8-4. Adding the Affinity Data.

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

Figure 8-5. Generating Map.

13. Next to Map topology type, choose Optimal.
14. 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.

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

18. Click Settings.
19. In the Simulation Parameters section, change the Simulation time for the Complex stage to 20.0.
20. 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.

21. Click OK.

Figure 8-8. Changing 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.

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

Figure 8-9. Opening the FEP+ output.

25. Find and open the FEP+ panel from the Tasks.
26. For Import structures or perturbation map from, choose File.
27. Click Browse.
28. Choose FEPMapperJob_5ANT_out.fmp.
29. Click Next.

Figure 8-10. The Export 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.

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

Figure 8-11. Viewing details for each edge.

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

31. In the FEP+ panel, go to the Analysis tab.
32. Hover your mouse pointer over one of the cells labeled Bad to see more details.
33. 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.