Forming RNA – Ligand Interactions with Ligand Designer

Tutorial Created with Software Release: 2025-3
Topics: Hit-to-Lead & Lead Optimization, Medicinal Chemistry Design, Small Molecule Drug Discovery
Products Used: Ligand Designer, Maestro

Tutorial files

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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 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:

 

This tutorial demonstrates how to introduce chemical modifications to the ligand to optimize interactions with the RNA receptor using the Ligand Designer tool in Maestro.

 

Tutorial Content

  1. Introduction to Ligand Designer

  1. Creating Projects and Importing Structures

  1. Prerequisites for Using the Ligand Designer

  1. Modifying the Ligand with Ligand Designer

  1. Conclusion and References

  1. Glossary of Terms

1. Introduction to Ligand Designer

Optimizing small molecules to enhance binding affinity, improve selectivity and achieve desirable drug-like properties is an important step in drug discovery. This process requires the understanding of ligand structure and its surrounding environment. Pharmacophore models such as hydrogen bond donors and acceptors, aromatic rings, hydrophobic regions, etc. and precise mapping of stable and unstable water molecules within the binding site can guide structural modifications.

Ligand Designer allows you to examine the non-covalently bound ligand and its interactions with the receptor to modify the ligand’s structure and get rapid feedback on your ideas. Libraries of fragments are available to enumerate possible modifications to the ligand. The resulting structures are automatically docked to the receptor, and the structures that do not form a good pose are eliminated. You can also use the pharmacologically relevant properties which are automatically calculated to filter out ligands that do not meet specified criteria, such as the Lipinski filter. For detailed information, please refer to the Ligand Designer panel documentation.

This tutorial uses the prepared 6DN3 RNA structure, which is a flavin mononucleotide (FMN) riboswitch bound to a flavin analog ligand BRX1555. If you are following along with a different structure, please make sure that your structure is prepared as shown in the Preparing Nucleic Acid Structures tutorial. If you prefer an example using a protein-ligand system, see the Forming Protein-Ligand Interactions with the Ligand Designer tutorial.

In this tutorial, you will learn how to identify modifiable regions of a ligand, introduce chemical modifications, and assess the resulting changes in binding interactions.

2. Creating Projects and Importing Structures

At the start of the session, change the file path to your chosen Working Directorythe location where 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 saved, the project is automatically saved each time a change is made.

Structures can be built in Maestro or can be imported using File > Import Structures (or drag-and-dropped), 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 2-1. Change Working Directory option.

  1. Go to File > Change Working Directory.
  2. Find your directory, and click Choose.
  3. Pre-generated 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/liganddesigner_nucleic_acid.zip
  4. 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.

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

Figure 2-3. Importing the prepared structure.

 

  1. Go to File > Import Structures.
  2. Navigate to and find RNAprep_6DN3-out.maegz.
  3. Click Open.
    • A new entry titled 6DN3 - prepared is added to the Entriesa simplified view of the Project Table that allows you to perform basic operations such as selection and inclusion and a banner appears confirming successful import of the structure.

Note: Imported structures in Maestro 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 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 in the Entriesa simplified view of the Project Table that allows you to perform basic operations such as selection and inclusion by default. 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.

3. Prerequisites for Using the Ligand Designer

The Ligand Designer uses core-constrained Glide docking to determine ligand poses. As such, all requirements and recommendations regarding structure preparation are the same for Ligand Designer and Glide docking. You should use the Protein Preparation Workflow to identify and resolve common issues (see the Introduction to Structure Preparation tutorials for Proteins and Nucleic acids, respectively), as well as follow the Best Practices for Protein Preparation. Any solvents, ions, or other molecules present in the structure besides the ligand will be considered an immutable part of the receptor, so make sure you are working with a clean and dry structure.

Additionally, keep in mind that the docking is performed with a rigid receptor, and so using a ligand-bound structure (experimental or computationally predicted) and understanding your target’s flexibility are highly recommended.

4. Modifying the Ligand with Ligand Designer

In this section, you will examine the 6DN3 FMN riboswitch receptor pharmacophore sites such as donors, acceptors, rings, hydrophobic groups to modify the flavin analog BRX1555 ligand to optimize its binding by performing R-group enumeration using the Ligand Designer panel.

Please note that the Ligand Designer tool is used here solely to demonstrate the steps involved in performing R-group enumeration and visualizing the modifications. The newly generated ligand variants are not intended for use in binding affinity predictions, as the poses generated by the Ligand Designer are not accurate enough to directly be used in FEP+ calculations.

4.1 Perform R-group enumeration

Figure 4-1. Splitting the prepared structure.

 

First, you need to isolate the ligand and receptor and remove the crystallographic water molecules. Note that ions present in the originally published structure were already removed before preparation.

 

  1. Right-click on 6DN3 - prepared and choose Split > Into Ligands, Water, Other.
    • The original entry is split into individual entries.

Figure 4-2. Renaming and including the split entries in the Workspace.

 

  1. Double-click the 6DN3 - prepared_protein and rename it to 6DN3 - prepared_RNA_apo.
  2. Includethe entry is represented in the Workspace, the circle in the In column is blue 6DN3 - prepared_RNA_apo.
  3. Ctrl+Click (Cmd+Click) to includethe entry is represented in the Workspace, the circle in the In column is blue 6DN3 - prepared_ligand and 6DN3 - prepared_RNA_apo in the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed.

Figure 4-3. The Ligand Designer in Tasks.

  1. Go to Tasks > Browse > Lead Optimization > Ligand Designer.

Figure 4-4. Analyzing the Workspace.

 

When initialized, the Ligand Designer analyzes the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed and applies visualization settings which make it easy to focus on the ligand and its immediate environment.

 

  1. In the Ligand Designer panel, click Analyze Workspace.
    • A new group named Ligand Designer is added to the Entriesa simplified view of the Project Table that allows you to perform basic operations such as selection and inclusion.
    • The Workspacethe 3D display area in the center of the main window, where molecular structures are displayed now has a single ligand and a blue-colored cloud surrounding it which represents the empty space between the ligand and the receptor.  

Note: If you want to retain your preferred style settings, you can uncheck the Adjust view and style when analyzing option.

You can customize what to focus on by using the toggles in the Display section. See the panel documentation for detailed explanations of the different options. You can also use the Cog next to the Workflows selector to customize the styling of the different Ligand Designer-specific workspace elements (such as the colors of the growth spaces, or how many potential interaction points should be visible).

Figure 4-5. Forming ligand-receptor interactions and displaying pharmacophore features on the receptor.

 

You can try forming an interaction with a nearby residue. First, you need to display the pharmacophore representations of the receptor interaction points.

 

  1. In the Display section, toggle on Ligand-Receptor Interactions.
    • The pharmacophore features on the RNA receptor that could potentially interact with the ligand are displayed.
    • You can hover over the interaction markers to see the corresponding receptor residues in the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed.

 

Note: It may take a few seconds for the Ligand-Receptor Interactions to be available.

Figure 4-6. Selecting the pharmacophore feature on G 11 (Donor) residue.

 

The X:G 11 residue presents an accessible hydrogen bond donor region. The phenyl moiety of the ligand offers an opportunity for enumeration without disrupting the ligand’s core structure.

 

  1. Select the pharmacophore feature on G 11 (Donor) residue in the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed.
    • A banner appears with a prompt to examine how the ligand side chain interacts with the receptor.
  2. Click OK in the banner.
    • A banner appears with a prompt to pick an arrow for specifying the enumeration site.
    • The available side chains are marked with colored arrows.

Figure 4-7. Specifying the enumeration site.

You can now choose a vector on the ligand from which to grow towards the interaction point.

 

  1. Click on the marked yellow-colored  arrow on the phenyl moiety of the ligand.
    • The Modify lead banner appears.

Figure 4-8. Running the enumeration job.

  1. Click Enumerate in the banner.
    • This job will take a few seconds.
    • A banner appears and 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 once the enumeration is finished and the ligands are successfully docked.

The cog (settings button) in the Modify lead banner allows you to control how the ligand variants are generated through R-group enumeration. You can choose between the default R-group library or a Custom library (e.g., solubilizing groups, hydrogen bond donors/acceptors, rings, etc.). You can also specify your own curated R-group library by choosing the “File” option in the Custom R-group library menu, or create your library using the R-Group Creator panel. If the enumeration yields fewer ligands than desired, enabling the option to “Include a diverse library” ensures additional chemically diverse variants are added until the specified threshold is met. You can also cap the total number of generated ligands to keep the enumeration manageable.

4.2 Visually analyze the enumerated ligands

Figure 4-9. Selecting the newly added group for the enumerated entries.

Ligand Designer creates Entry groups for the results of each workflow.

 

  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 newly added group for enumerated entries by clicking on the group heading.

There are various tools available to help you filter through the enumeration results. You can use the cog to adjust the properties included in the Multi-Parameter Optimization plot and MPO score, and use that to sort the Entries. As you step through the Entries, the properties shown in the Multi-parameter optimization section update as well. Sorting by clashes or good interactions can help you identify efficient ligands.

 

Note that the Ligand Designer intentionally does not show the docking score. Due to the underlying approximations of core-constrained, rigid-receptor docking with Glide, the docking score should not be used to rank-order congeneric ligands.

Figure 4-10. Sorting the enumerated entries by good interactions.

  1. In the Ligand Designer panel, under POST-PROCESSING, choose Sort by Good Interactions for Selected entries and click Sort.
    • The ligand variants are sorted by good interactions.

Figure 4-11. Visualizing the ligand variants.

  1. Includethe entry is represented in the Workspace, the circle in the In column is blue the ligand variants one at a time and visually analyze them in the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed.

 

Optional: Click the Star to mark ideas as favorites while going through the results. You can group your favorites together in the project for additional review.

The newly introduced R-groups extend into the pocket near X:G11 residue and are engaged in favorable hydrogen-bonding interactions, thereby improving the ligand-receptor binding.

Figure 4-12. Performing reaction based enumeration.

You can add further modifications to a ligand idea resulting from a previous enumeration. Includethe entry is represented in the Workspace, the circle in the In column is blue your favorite ligand idea and repeat the process to form an interaction with the hydroxy group on U 61.

 

  1. Select the pharmacophore feature on the U 61 (Acceptor) residue.
    • A banner appears.
  2. Click OK in the banner.
  3. Choose the marked purple-colored arrow and click Enumerate in the Modify lead banner.
    • This job will take ~5 minutes.
    • A banner appears and a new group is added to Entriesa simplified view of the Project Table that allows you to perform basic operations such as selection and inclusion once the job is completed.

The purple-colored arrows designate connection points where reaction-based enumeration can be performed, which considers established synthetic routes for generating the substituents. Read more on reaction-based enumeration in the Pathfinder documentation.

 

For examples of applying other Ligand Designer workflows, see the Forming Protein-Ligand Interactions with the Ligand Designer and WaterMap-Guided Lead Optimization with the Ligand Designer tutorials.

5. Conclusion and References

In this tutorial, you learned how to use the Ligand Designer panel to perform R-group enumeration to introduce chemical modifications in the ligand and improve its binding to the receptor.

Once you have a set of ligands, you can export them into other workflows, for example, send them to the FEP+ panel to evaluate the relative binding free energies for the new ligands and the input ligands as described in the Potency Predictions for RNA-Binding Small Molecules Using RB-FEP tutorial. See the further learning section below or the Oligonucleotide Modeling learning path for additional resources.

6. 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

Recent actions - This is a list of your recent actions, which you can use to reopen a panel, displayed below the Browse row. (Right-click to delete.)

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 where files are saved

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