Using IFD-MD on a covalently-bound ligand

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

Tutorial files

1.3 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 displayed.the 3D display area in the center of the main window, where molecular structures are displayed
Abstract:

 

In the same manner as noncovalent induced fit docking, sometimes prediction of the proper binding pose for a covalent inhibitor requires coincident binding site structural changes to accommodate the proper ligand binding pose. In these situations, rigid receptor docking falls short of the aim to predict correct covalent ligand binding modes.

 

The example addressed in this tutorial is the docking of a potent covalent inhibitor, zanubrutinib (PDB: 6J6M), into the structure of Bruton’s tyrosine kinase (BTK; PDB: 5BQ0), a protein target investigated for treatments against mantle cell lymphoma, multiple sclerosis, and rheumatoid arthritis. In this example, we take advantage of the known binding conformation of zanubrutinib co-crystallized with BTK (PDB: 6J6M). In order to predict the correct binding pose for zanubrutinib, the Lys430 side chain must be moved out of the way to accommodate the phenoxyphenyl group of the inhibitor. Due to this induced fit binding, it is expected that rigid receptor docking will not be able to predict the correct binding pose.  

Tutorial Content

  1. Introduction to IFD-MD

  1. Creating Projects and Importing Structures

  1. Preparing a covalently-bound ligand complex for IFD-MD

  1. Setting up and submitting the CovDock IFD-MD Job

  1. Visualization and analysis of IFD-MD results

  1. Conclusion and References

  1. Glossary of Terms

1. Introduction to IFD-MD

Many structure-based drug design methods, including free energy perturbation (FEP+), require accurate, atomic-level detail structures of the target protein complexed with a member of the ligand series being explored to perform optimally; consequently, the domain of applicability of structure-based drug design (SBDD) is limited by the availability of high-resolution crystal structures. Even when the exact protein-ligand complex structure needed for design work is not available, accurate atomic-level detail structures are often available for similar systems. There is often a significant lag between the identification of a hit and the structural-enablement of drug discovery work around that hit. A new ligand subseries sometimes results in a changed binding mode. IFD-MD enables structure-based drug discovery before the relevant co-crystal structure is available.

Often the only differences in protein structure between the available structure and the one that is needed for SBDD are the movements of a couple of side chains or a small loop motion. However, these small changes can have a large impact on the accessible ligand binding modes. IFD-MD uses a combination of docking algorithms, water thermodynamics, empirical scoring functions, implicit solvent force field energies, and explicit solvent metadynamics trajectories to explore the motions of the target protein and simultaneously determine their relative energetics. This technology makes it possible to create accurate “first looks” at the protein-ligand interactions for novel active compounds before a crystal structure is solved, and even allows for accurate structures of protein-ligand binding when starting from homology models. IFD-MD integrates multiple Schrödinger applications into a single solution for predicting binding poses, operating as a series of coupled CPU and GPU jobs:

 

Figure 1. IFD-MD Subjobs. Each dashed vertical line is a checkpoint. The job can be restarted from any checkpoint.

Initial Pose Generation (Phase, Prime, Glide)

Initial pose generation is done by first pharmacophore docking your ligand of interest (target ligand) onto the ligand of an existing holostructure ligand (template ligand). This ligand-based docking initially ignores clashes to the receptor. Pharmacophore docking generates thousands of conformations of your target ligand. For each conformation, clashes with the receptor are resolved using Prime to produce potentially a unique receptor conformation for each ligand conformation. The new receptor conformations are then re-docked using Glide to create an initial induced fit ligand receptor complex. For more details, please consult the references mentioned at the end of the tutorial.

Molecular Dynamic Stages

Concerted ligand-receptor changes are accomplished through short time scale (500 ps) MD simulations. The short MD time-scale also includes the placement of explicit water molecules, necessary for inclusion in scoring with Glide WS. The stability of a pose is evaluated using multiple metadynamics (MtD) calculations. Poses with more consistent conformations score more favorably. Poses with greater conformational variability score less favorably. IFD-MD incorporates this MtD stability as a component of its scoring function. A final 100 ns MD simulation is performed on the top two IFD-MD structures.

Note: Drastic backbone changes, i.e. DFG-in to DFG-out, are beyond the scope of IFD-MD predictions.

For more information about IFD-MD, please see the IFD-MD panel documentation as well as the IFD-MD Best Practices document.

2. 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 Directory 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. table is located to the left of the Workspace. 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 to start Maestro.

Figure 2-1. 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/covalent_ifd-md.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 2-2. Saving the project.

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

3. Preparing a covalently-bound ligand complex for IFD-MD

Structures obtained from the PDB, vendors, and other sources often lack necessary information for immediately performing modeling-related tasks. Typically, these files are missing hydrogens, partial charges, side chains, and/or whole loop regions. In order to make these structures suitable for modeling tasks, such as IFD-MD, we will use the Protein Preparation Workflow to resolve these issues. Please see the Introduction to Structure Preparation and Visualization tutorial and the Best Practices for Protein Preparation document for further information on using the Protein Preparation Workflow. Here, we will use the Protein Preparation Workflow default settings.

Similarly, ligand files can be sourced from numerous places, such as vendors or databases, often in the form of 1D or 2D structures with unstandardized chemistry. LigPrep can convert ligand files to 3D structures, with the chemistry properly standardized and extrapolated, ready for use in modeling tasks. Even though we will be using a ligand from a crystal structure for this IFD-MD calculation, we will perform this step as part of our recommended best practices.

3.1 Preparing proteins and ligands for use in IFD-MD

 

Figure 3-1. Downloading the template receptor.

  1. Go to File > Get PDB.
    • The Get PDB File panel opens.
  2. For PDB IDs write 5BQ0.
  3. For Chain name write A.
  4. Click Download.
    • The structures are added to the Entriesa simplified view of the Project Table that allows you to perform basic operations such as selection and inclusion..
    • 5BQ0 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..

Figure 3-2. Opening the Protein Preparation Workflow panel.

  1. In the Favorites Toolbar, click Protein Preparation Workflow.
    • The Protein Preparation Workflow panel opens.

 

Note: We will use the default settings for the Protein Preparation Workflow here. See the Protein Preparation Workflow user manual for more information on the setting options.

Figure 3-3. Loading the 5BQ0 structure into the Substructures tab of the Protein Preparation Workflow.

  1. Go to the Substructures tab and click Load Workspace Entry.
    • All substructures from the 5BQ0 PDB file are now broken down by whether they are protein, water, or ligands/metal/other.

Figure 3-4. Remove crystallographic artifacts.

  1. Ctrl+Click (Cmd+Click) to select the DMS and EDO entries in the Ligands, Metals, Other table.
  2. Click Delete from Entry.
    • Crystallographic artifacts from the template 5BQ0 structure have been removed.

Figure 3-5. Customize Protein Preparation Workflow settings.

  1. Go back to the Preparation Workflow tab.
  2. In the Preprocess section, select More Options and uncheck Generate het states (with Epik).
  3. In the Optimize H-Bond Assignments section, click Settings and check Minimize hydrogens of altered species.
  4. In the Minimize and Delete Waters section, click Settings and select Optimize hydrogens only and choose to delete waters 0 Å from the ligands(hets).

Figure 3-6. Running the preparation job.

  1. Next to Job name, type proteinprep_5BQ0.
  2. Click Run.
    • This job takes a few minutes to complete.
    • Once the job is completed, 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..  

3.2 Importing and preparing the target ligand structure

Figure 3-7. Download the 6J6M structure from the PDB.

  1. Go to File > Get PDB.
    • The Get PDB File panel opens.
  2. For PDB IDs, type 6J6M.
  3. For Chain name write A.
  4. Click Download.
    • The structure is added to the Entriesa simplified view of the Project Table that allows you to perform basic operations such as selection and inclusion. and 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..

Figure 3-8. Choosing to split the structure to isolate the ligand.

  1. Right-click the 6J6M entry and choose Split > Into Ligands, Water, Other.
    • Three 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. table.

 

 

 

 

 

Since the ligand is covalently bound to the receptor, splitting the entries into individual components simply removes the bond between zanubrutinib and the reactive side chain, but it does not re-generate the correct bond order for the ɑ,ꞵ-unsaturated carbonyl.

Figure 3-9. Increasing the bond order for the ɑ,ꞵ-unsaturated carbonyl.

  1. Includethe entry is represented in the Workspace, the circle in the In column is blue. only 6J6M_ligand in the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed..
  2. In the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed., ctrl+click (cmd +click) to select the two terminal carbon atoms of the short aliphatic chain.
  3. Right-click on the bond between the two atoms and select Increase Bond Order.
    • The bond order is increased.

Figure 3-10. Preparing the ligand with LigPrep.

  1. Find and open LigPrep from Tasks.
  2. For Use structures from, choose Workspace (1 entry).
  3. For Ionization, check Include original state.
  4. In the Stereoisomers section, choose Determine chiralities from 3D structure for Computation.
  5. Change the Job name to 6J6M_ligand_ligprep.
  6. Click Run.
    • This job takes a few minutes.
    • Once the job is completed, 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..

4. Setting up and Submitting the CovDock IFD-MD Job

With our prepared ligands and protein, we are now ready to set up and submit the IFD-MD calculation. For compute intensive jobs such as IFD-MD, you may prefer to write out the calculation input files and transfer them to a remote cluster for running. Here, we show how to submit the calculation from your local computer to a remote cluster using Job Server.

Figure 4-1. Including and selecting the input structures.

  1. In the Entriesa simplified view of the Project Table that allows you to perform basic operations such as selection and inclusion., includethe entry is represented in the Workspace, the circle in the In column is blue. the 5BQ0 - prepared and select 6J6M_ligand entry (from the LigPrep output group).    

Figure 4-2. Loading the input structures into the panel.

  1. Find and open IFD-MD from Tasks.
  2. In the IFD-MD panel, under Docking, choose Covalent.
  3. Next to Target Ligand, choose Project Table (1 selected) and click Load.
    • The 6J6M_ligand structure is loaded into the panel.

 

Note: Template ligand is permitted to be either covalently-bound or noncovalently-bound.

 

  1. Next to Template complex from Workspace, click Load.
    • The 5BQ0 structure is loaded into the panel.

 

The ligand(s) text box is auto populated with the label A:4US (701) since it is the only ligand identified by Maestro from the template complex. If the text box is not auto populated or is incorrect, you can click the “Pick” box and subsequently click on any template ligand atom in Maestro. Selection will be expanded to the encompassing residue for the picked atom.

Figure 4-3. Choose the reactive residue.

  1. In the IFD-MD panel, switch to the Reaction tab.
  2. Select Pick next to Reactive Residue.
    • A banner prompts you to pick the reactive residue.
  3. In the Hierarchy, search 481.
  4. Select CYS 481 from the results.
  5. Click the Fit view to selected atoms button.
    • The Workspacethe 3D display area in the center of the main window, where molecular structures are displayed. zooms to the CYS 481 residue.
  6. In the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed., click on any atom of the CYS 481 residue.
    • CYS 481 is loaded as the reactive residue in the IFD-MD panel.

 

 

Figure 4-5. Choosing the reaction type.

  1. Next to Reaction type, choose Michael Addition.

 

Note: If the standard reaction type expected for the job is not included in the drop-down menu, double check the selected side chain and the selected target ligand.

 

  1. For Reaction site, choose Site #1.
    • The selected reaction site will be highlighted in pink in the 2D viewer.

 

The list of standard reactions in the dropdown menu is pre-populated based on the side chain and ligand identities. Only reactions that have SMARTS pattern matches for both the side chain and ligand are included in the dropdown menu.

 

If a chiral center is formed when the reaction is undergone you will then be prompted to include the desired chirality as part of the reaction settings.

 

The full list of standard reactions can be found in the table below:

If you would like to create a custom reaction file, please follow the instructions here.

Figure 4-6. Choose the CPU and GPU hosts.

  1. Click the Job settings (cog) icon.
  2. Choose the Driver Host and the number of processors (we would recommend 2+, if possible).
  3. Choose the CPU subhost and number of processors.
  4. Choose the GPU subhost and number of processors.
  5. Optional: click Run
    • This job is time-intensive.
    • You will use pre-generated results to save time.

Figure 4-7. Writing the input files.

Note: If preferred, you can write out the input files for transfer to a remote cluster to run this job. Be sure that the appropriate hosts are chosen in the Job Settings.

5. Visualization and analysis of IFD-MD results

The quality of the results for the docking of zanubrutinib (PDB:6J6M) into BTK (PDB: 5BQ0) can be assessed by taking advantage of the crystal structure of zanubrutinib co-crystallized with BTK (PDB: 6J6M). Generally, the results from a retrospective IFD-MD job are considered successful if at least one of the top two ligands poses has a sub 2.5Å heavy-atom ligand RMSD relative to the crystal structure.

Figure 5-1. Importing the pre-generated IFD-MD output and pre-prepared 6J6M structure for reference.

  1. Navigate to File > Import Structures and choose btk_5bq0_btw_6j6m-out.maegz and 6J6M-prepared.mae.
  2. Click Open.

Figure 5-2. Using the Binding Mode Comparison Preset.

  1. Ctrl+Click (Cmd+Click) to includethe entry is represented in the Workspace, the circle in the In column is blue. the top 6J6M_ligand entry in the btk_5bq0_btk_6j6m-out group and the 6J6M-prepared entry.
  2. Go to Presets > Binding Mode Comparison.

Figure 5-3. Tiling the Workspace with the IFD-MD pose (green) next to the crystal structure (grey).

  1. Use Ctrl+L (Cmd+L) to tile the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed..
    • You can now view the crystal structure and the top-ranked IFD-MD pose side-by-side in the tiled Workspacethe 3D display area in the center of the main window, where molecular structures are displayed..

 

The result of running CovDock IFD-MD led to a 0.4Å ligand RMSD relative to the crystal structure (see Figure 5-3). The success of CovDock IFD-MD is partially due to the sampling of the Lys430 side-chain (shown in orange below) which allowed it to move out of the binding site and open up a new subpocket for the phenyl substituent to fill and interact with PHE 540.

 

Figure 2. Best ranked Covalent IFD-MD output with LYS 430 and PHE 640 shown.

 

This is in contrast with the use of a more static receptor structure, where running Covalent Docking produces a 6.2Å pose, which was ranked 5th. The reason for this discrepancy is that in order to produce the correct docking pose, the Lys430 side chain in the receptor from PDB: 5Q60 must move out of the ligand binding site.

 

Figure 3. Comparison of the predicted binding modes when using Covalent IFD-MD (left, green) and standard rigid receptor CovDock (right, pink).

 

When covalent docking with a rigid receptor is used, the LYS 430 cannot be moved and the ligand binding pose must be reoriented so as to avoid steric clashes with the lysine. This leads to a very different binding conformation. In the image above, the green structure is the crystal structure and the pink structure is the best result from running rigid receptor covalent docking, using the most enhanced sampling mode which includes very modest side chain refinement. LYS 430 is included in orange for reference.

6. Conclusion and References

In this tutorial you learned how to use IFD-MD’s covalent docking mode to dock zanubrutinib (from PDB:6J6M) into a different BTK crystal structure (PDB: 5BQ0). We used a Michael Addition reaction to form the covalent bond between the ligand and the protein, and in the end the best pose (as determined by the IFD-MD scoring function) was found to have a 0.8 Å ligand RMSD relative to the crystal structure.

7. Glossary of Terms

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

Favorites Toolbar - buttons for tasks designated as favorites in the Task Tool. You can add panels to your Favorites Toolbar by checking the star icon beside the panel name in Tasks.

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.