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

Figure 2-1. Change Working Directory option.

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/sbvs_phase.zip
5. 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 2-2. Save Project panel.

8. Go to File > Open Project.
9. Choose lkha4_pharmacophore.prjzip.
10. Click Open.
11. In the Save scratch project dialog box, click OK.
12. Go to File > Save Project As.
13. Change the File name to Phase_tutorial.
14. Click Save.
    • The project is now named Phase_tutorial.prj.
    • Structures are shown in the Entriesa simplified view of the Project Table that allows you to perform basic operations such as selection and inclusion.

Note: Please see 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 3-1. Develop Pharmacophore Hypothesis option in the Ligand-Based Virtual Screening.

1. Confirm that the 3CHP_prepared is 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.
2. Double-click Presets.
   • The structure is rendered in the default custom preset and the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed zooms to the ligand.
3. Go to Tasks > Browse > Ligand-Based Virtual Screening > Develop Pharmacophore Hypothesis.
   • The Develop Pharmacophore Model panel opens.

Figure 3-2. Creating a complex hypothesis using E-Pharmacophores.

4. Under Create pharmacophore model using, choose Receptor-ligand complex (Workspace).
5. For Method, choose Auto (E-Pharmacophore).
6. Under Choose ligand, click A:4BO (901).
7. Click the Hypothesis Settings button.
   • The Hypothesis Settings dialog box opens.

 

Figure 3-3. The Features tab in the Hypothesis Settings dialog box.

8. In the Features tab, choose Donors as vectors.

Figure 3-4. The Excluded Volumes tab in the Hypothesis Settings dialog box.

9. Go to the Excluded Volumes tab.
10. Check Create receptor-based excluded volumes shell.
11. Click Save.

Figure 3-5. Creating hypothesis.

12. In the Develop Pharmacophore Model panel, change the Job name to lkha4_epharm_exvol.
13. Click Run.
    • This job takes a few minutes.
    • A banner appears in the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed when the job has been incorporatedonce a job is finished, output files from the working directory are added to the project and shown in the Entry List and Project Table.

Figure 3-6. E-Pharmacophore overlaid  with 3CHP prepped complex.

14. Ctrl+Click (Cmd+Click) to includethe entry is represented in the Workspace, the circle in the In column is blue lkha4_epharm_exvol and 3CHP_prepared in the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed.
    • The generated E-Pharmacophore is now overlaid in the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed with the input protein-ligand complex.

Figure 3-7. Creating a complex hypothesis manually.

Several studies have noted the presence of hydrogen-bonding between the TRP 311 residue backbone and the bound ligand. Since that feature is not present in the previously generated pharmacophore hypothesis, you can add it through a manual hypothesis.

15. In the Develop Pharmacophore Model panel, choose Manual.

Note: There are some features that were not included in the E-Pharmacophore that can be chosen when generating a Manual Hypothesis. This is due to a difference in cutoff between the two tools.

16. Under Show features, drag the slider right to More.
    • Features D3, N4, P5, and R6 are shown.
17. Click Hypothesis Settings.
    • The Hypothesis Settings dialog box opens.

Figure 3-8. The Hypothesis Settings dialog box.

18. Check Create receptor-based excluded volumes shell.
19. Click Save.

Note: Selecting Show inverse features from receptor generates additional features based on idealized geometries of specific interactions and hydrophobic features of the binding site.

Figure 3-9. The Interactions panel.

20. In the Workspace Configuration Toolbar (bottom right), right-click Interactions Toggle.
    • The Interactions panel opens.
21. Next to Non-covalent bonds, choose Ligand-receptor.
22. Toggle on Aromatic H-bonds.
    • Aromatic H-bonds are shown in the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed.
23. Under Contacts/Clashes, toggle off Bad.
    • Bad clashes are hidden in the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed.

 

Figure 3-10. Selecting TRP 311 residue.

24. In the Hierarchy, search for TRP 311 using the search toggle.
25. 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 TRP 311.
    • The TRP 311 residue is selected in the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed.
26. Click Fit view to selected atoms.
    • The Workspacethe 3D display area in the center of the main window, where molecular structures are displayed zooms to TRP 311.

Note: You may also double-click TRP 311 in the Hierarchy to zoom to the selected residue.

 

Figure 3-11. Adding a Donor in Workspace.

27. In the Develop Pharmacophore Model panel, under Choose features, check Add and choose Donor in Workspacethe 3D display area in the center of the main window, where molecular structures are displayed.
    • A banner appears prompting you to pick an atom.

Figure 3-12. Selecting atom H20 on the ligand.

28. 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 atom H20 on the ligand, which makes an aromatic H-bond with the backbone of TRP 311.
    • A donor vector D7 appears in the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed.
    • D7 is checked in the Choose features box in the Develop Pharmacophore Model panel.

Figure 3-13. Manually repositioning the D7 vector.

29. In the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed, right-click the D7 vector.
    • The Edit Feature D7 dialog box opens.
30. Check Manually reposition feature.
31. Click and drag the scroll wheel (Shift-click and drag on a Trackpad) to reposition the vector along the hydrogen bond.
32. Click OK.

Note: See the Custom Mouse Actions Panel help topic for further information on mouse actions.

Figure 3-14. Creating manual hypothesis.

33. In the Develop Pharmacophore Model panel, under Choose features, check all the features (D3, D7, N4, P5, R6).
34. Change the Job name to lkha4_manual.
35. Click Create.
    • This job takes a few seconds.
    • A banner appears in the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed confirming the job has been successfully imported.
    • A new entry titled lkha4_manual is added to the Entriesa simplified view of the Project Table that allows you to perform basic operations such as selection and inclusion.
36. Close the Develop Pharmacophore Model panel.  

Figure 3-15. Manual Pharmacophore overlaid with the 3CHP prepared complex.

37. Ctrl+Click (Cmd+Click) to includethe entry is represented in the Workspace, the circle in the In column is blue lkha4_manual and 3CHP_prepared in the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed.
    • The generated E-Pharmacophore is now overlaid in the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed with the input protein-ligand complex.

Figure 3-16. Showing hypothesis feature properties in the Workspace.

Note: For ligand screening, it is at times desirable to broaden or narrow the types of moieties a particular pharmacophore feature is allowed to match.

38. Includethe entry is represented in the Workspace, the circle in the In column is blue lkha4_manual in the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed.
39. Next to lkha4_manual, click the H.
40. Choose Show > Feature Properties.
    • Properties of each feature are displayed in the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed.

Note: Excluded volumes can be edited manually. As a pedagogical practice, we will demonstrate adding and removing excluded volumes in the next steps.

Figure 3-17. Selecting HIP 139 residue.

41. Ctrl-click (Cmd-click)  to includethe entry is represented in the Workspace, the circle in the In column is blue lkha4_manual and 3CHP_prepared in the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed.
42. In the Hierarchy, search for HIP 139 using the Search toggle.
43. 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 HIP 139.
44. Click Fit view to selected atoms.
    • The Workspacethe 3D display area in the center of the main window, where molecular structures are displayed zooms to HIP 139 residue.

Figure 3-18. The Manage Excluded volumes option.

45. Next to lkha4_manual, click the H.
46. Choose Show > Manage excluded Volumes.
    • Manage Excluded Volumes Dialog box opens.

Figure 3-19. Deleting excluded volumes for HIP 139 residue.

47. Click Selected Atoms.
    • Excluded volumes are added for atoms in HIP 139.
    • Information is added to the bottom of the table.
48. Scroll to the bottom of the table 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 last excluded volume.
    • 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 excluded volume is highlighted in the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed.
49. Click Delete.
    • The highlighted excluded volume is deleted from the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed.
50. Click Close.

Figure 3-20. Adding Reference ligands to the Workspace for both included entries.

Merging hypotheses allows you to develop multiple models and choose common/desired features manually.

 

51. Ctrl+Click (Cmd+Click) to includethe entry is represented in the Workspace, the circle in the In column is blue lkha4_epharm_exvol and lkha4_manual in the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed.
52. For each includedthe entry is represented in the Workspace, the circle in the In column is blue entry, click the H and choose Show then Reference ligand
    • Reference ligands are added to the Entriesa simplified view of the Project Table that allows you to perform basic operations such as selection and inclusion and are includedthe entry is represented in the Workspace, the circle in the In column is blue and fixed in the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed.

Figure 3-21. Creating merged hypotheses for the chosen features.

53. Go to Tasks > Browse > Ligand-Based Virtual Screening > Develop Pharmacophore Hypothesis.
    • The Develop Pharmacophore Model panel opens.
54. For Create pharmacophore model using, choose Merged hypotheses (Workspace).
55. For Choose features, from lkha4_epharm_exvol, choose R6 and R7.
56. For Choose features, from lkha4_manual, choose D3, N5, P6, and XVols.

 

Note: The D3 feature is the donor that we added manually. The R6 feature in the E-Pharmacophore aligns well with the R7 from the manual pharmacophore, so only one of them needs to be added to the merged hypothesis.

 

57. Change the Job name to lkha4_merge.
58. Click Create.
    • This job takes a few seconds.
• A banner appears in the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed confirming the job has been successfully imported.
• A new entry titled lkha4_merge is added to the Entriesa simplified view of the Project Table that allows you to perform basic operations such as selection and inclusion.
59. Close the Develop Pharmacophore Model panel.

Figure 4-1. Entries selection and inclusion.

1. In the Entriesa simplified view of the Project Table that allows you to perform basic operations such as selection and inclusion, 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 DUD-E_Actives and DUDE_Inactives groups.
2. Includethe entry is represented in the Workspace, the circle in the In column is blue lkha4_merge in the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed.

 

Figure 4-2. The Phase Ligand Screening panel.

3. Go to Tasks > Browse > Ligand-Based Virtual Screening > Ligand Screening.
   • The Phase Ligand Screening panel opens.
4. For Ligands to screen, choose Project Table (selected entries).
   • 215 ligands will be screened.
5. Click Add Hypothesis.
6. Choose Workspace.
   • lhka4_merge is added to the table.
7. For the lkha_merge hypothesis, click the Matches column and choose 3 of 5.
   • Ligands must satisfy at least 3 features of each hypothesis.
8. Click the Screening Settings button.
   • The Screening Settings dialog box opens.

Figure 4-3. Adding features to ligand screening.

9. Go to the Groups tab.
10. Check Pick features.
    • A banner appears in the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed prompting you to pick features to be grouped.
11. Select features D1 and R5 in the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed.
12. Click Create Group.
    • The table is populated.

Note: Any ligand that does not match at least one of the grouped features will not be returned in the screen, even if it matches the desired 3 of 5 total features.

13. Click Save.

Figure 4-4. Running the Phase Ligand Screening job.

13. Change the Job name to lkha4_screen.
14. Click Run.
    • This job takes ~1 minute.
    • A banner appears in the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed confirming the job has been incorporated.
    • A new group lkha4_screen is added to the Entriesa simplified view of the Project Table that allows you to perform basic operations such as selection and inclusion.
15. Close the Phase Ligand Screening panel.

Figure 4-5. Visualizing the ligand screening results with the corresponding hypothesis.

16. 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 lkha4_screen group by clicking on the group heading.
17. Double-click the In circle of lhka4_merge to fix the hypothesis in the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed.
18. Includethe entry is represented in the Workspace, the circle in the In column is blue the top ligand.
19. Click Style selected entries (paint brush icon) and choose ball-and-stick representation.
20. Use the right and left arrow keys to step through the ligands.

Figure 4-6. The Show Property option in the Change table settings menu.

21. At the top right corner of 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 (three vertical dots) and choose Show Property.
    • The Show Properties in Table dialog box opens.

 

Figure 4-7. Adding properties to the table.

22. Click Choose.
23. Search for and select PhaseScreenScore and Matched Ligand Sites from the list.
24. Click OK.
    • The properties are added to the table.

 

 

Figure 4-8. Updated table with Matched Ligand Sites and PhaseScreenScore.

25. Hover over the Matched Ligand Sites circles to see what features are present.

 

Note: Only 46 of the 215 screened ligands were returned following the Phase Ligand Screen due to the matching criteria. Of those returned, only 6 were inactives, and those had low Phase Screen Scores relative to the rest of the dataset.

 

Please note that your results may vary slightly if you are using a different version of the software.