Homology Modeling of Protein-Ligand Binding Sites with IFD-MD

Tutorial Created with Software Release: 2023-2
Topics: Small Molecule Drug Discovery, Structure Prediction & Target Enablement
Products Used: IFD-MD

Tutorial files

10 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

 

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:

 

In this tutorial you will learn how to create a homology model of TYK2, including a bound ligand, using the structure of JAK3 as a starting point. Then you will compare this homology model with the crystal structure for TYK2 bound to 4GIH ligand.

 

Tutorial Content

  1. Introduction

  1. Creating Projects and Importing Structures

  1. Creating Homology Models with Multiple Sequence Viewer/Editor

  1. Preparing the Homology Model with Protein Preparation Workflow

  1. Running IFD-MD Job

  1. Setting up FEP+ to Validate the Aligned Poses

  1. Analyzing FEP+ Job Results

  1. References

  1. Glossary of Terms

1. Introduction

Structure-based drug design (SBDD) is often hampered by the costs of obtaining a crystal structure. Here we present IFD-MD as a reliable and accurate solution for expanding the applicability of SBDD.

Most structure-based computational chemistry tools for small molecule design require crystal structures of the small molecule bound to the receptor of interest. What if there is no structure of the receptor of interest? Homology models can help understand the receptor, but this answers only part of the problem. Combining Prime Homology Modeling with IFD-MD can create models of the entire binding site where templates with >40% sequence identity are available.

Figure 1-1. IFD-MD homology modeling workflow.

Figure 1-2. Comparison of the experimental binding poses between a member of the congeneric series to predict in TYK2, ( grey in 3D, upper in 2D) and the experimental ligand binding pose of the ligand in the JAK2 structure ( 3ZC6) used as a template to predict the binding pose and the affinities with FEP+ ( pink in 3D, lower in 2D).

IFD-MD integrates multiple applications of the Schrödinger Suite into a single solution for predicting binding poses operating as a series of coupled CPU and GPU jobs:

 

Figure 1-3. IFD-MD Subjobs. Each dashed vertical line is a checkpoint. The job can be restarted from any checkpoint.

Initial Pose Generation (Phase, Prime, Glide)

The initial poses are generated by pharmacophore docking your ligand of interest, the target ligand, onto the ligand of an existing holo structure containing the template ligand. This ligand-based docking initially ignores clashes with 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 Dynamics Stages

Concerted ligand-receptor changes are accomplished through short time scale (500 ps) MD simulations. The short MD timescale also includes the placement of explicit water molecules which are necessary for the scoring with Glide WS. The stability of a pose is evaluated using multiple metadynamics (MtD) calculations. Poses with stable 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 if the structure appears unconverged after 500 ps.

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

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 Directorythe location that files are saved. using File > Import Structures, and are added to the Entry Lista 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 Entry Lista 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 2-2. 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/ifd-md_homology_modeling.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 2-3. Opening IFD-MD tutorial project file.

  1. Go to File > Open Project
  2. Select the ifd-md_homology_modeling.prjzip  and click Open
    • The pregenerated project opens as a 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 can be saved under a new name. Once a project is created, changes are saved automatically.

If you are unfamiliar with the Maestro GUI, please see the Glossary of Terms for the distinction 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 Entry List (and Project Table) and the row for the entry is highlighted. Project operations are performed on all selected entries. entries in the Entry Lista simplified view of the Project Table that allows you to perform basic operations such as selection and inclusion..

Figure 2-4. Save Project panel.

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

 

3. Creating Homology Models with Multiple Sequence Viewer/Editor

High quality alignments are essential for creating high-quality homology models. We will use the Multiple Sequence Viewer/Editor panel that allows these alignments to be rapidly analyzed interactively.

3.1     Preparing the Target Sequence

Figure 3-1. Launching Multiple Sequence Viewer/Editor panel.

  1. From TASKS in the toolbar, search for multiple sequence viewer
  2. In the displaying results, choose Multiple Sequence Viewer/Editor
    • Multiple Sequence Viewer/Editor panel opens  

Figure 3-2. Launching Build Homology Model panel.

  1. Below the main menu, click the View 1 tab
  2. Select Other Tasks from the Multiple Sequence Viewer/Editor toolbar and select Build Homology Model
    • The Build Homology Model panel opens

Figure 3-3. Importing the target sequence.

  1. Click Import Sequence
  2. Navigate to where you downloaded the tutorial files and choose tyk2.fasta
    • The tyk2 target sequence is loaded

Figure 3-4. Importing the PDB template.

  1. In the panel, go to File > Get PDB
  2. In PDB IDs, enter 3ZC6 and in the Chain name, type A in capital letter
  3. Click Download

Figure 3-5. Align the sequences.

  1. In the Build Homology Model panel click Run Alignment

Note: This is a simple alignment done with the MUSCLE method with the target TYK2 being the reference sequence as the first sequence  above the template 3ZC6 sequence.

You can click on the icon to view more information on this step.

3.2 Build the Homology Model

Figure 3-6. Choosing ligand template.

  1. In the Build Homology Model panel, find the “Include ligands & cofactors” portion of the workflow and click Choose
    • Choose Ligands and Cofactors panel opens
  2. Select VFC from the list
  3. Click OK

Figure 3-7. Selecting an energy-based homology model.

  1. In the Build Homology Model panel, find the “Change model settings” portion of the workflow and click View Settings
    • View Modeling Settings panel opens
  2. In the Modeling Method tab from the View Modeling Settings panel, select the Energy-based option
  3. Click OK

Figure 3-8. Generating the homology model.

  1. Name the Job homology_modeling_3ZC6
  2. Click Generate Model in the Build Homology Model panel
    • The job status at the bottom left of the Multiple Sequence Viewer/Editor panel changes to 1 homology modeling job in progress

When the homology modeling job finishes, a message will appear and the model will appear in your Entry Lista simplified view of the Project Table that allows you to perform basic operations such as selection and inclusion. as homology-out group. The job takes ~14 minutes. We will look at pregenerated results in the next step.

Figure 3-9. Importing pre-generated homology models.

  1. In Maestro, go to  File > Import Structures
  2. Navigate to your working directory and open the HomologyModeling_3ZC6 folder and then choose homology_modeling_3ZC6-out.mae from your calculation or the provided tutorial file
  3. Click Open
    • The homology model of TYK2 based on 3ZC6_A is included in your Workspacethe 3D display area in the center of the main window, where molecular structures are displayed.

 

Figure 3-10. The homology model of TYK2 based on 3ZC6_A.

The ligand is still the same ligand from the original JAK3 ligand even though we are interested in the TYK2 ligand.

  • Dark blue residues are conserved between template and target
  • Light blue residues are different between template and target sequence
  • Red residues are rebuilt

 

 

4. Preparing the Homology Model with Protein Preparation Workflow

Before proceeding with an IFD-MD job, you must prepare the protein and ligands. For more information regarding the effect of protein and ligand preparations and their protocols, please see Sastry, G.M.; Adzhigirey, M.; Day, T.; Annabhimoju, R.; Sherman, W., J. Comput. Aid. Mol. Des., 2013, 27(3), 221-234 (DOI: 10.1007/s10822-013-9644-8).

Figure 4-1. Prepare the homology model using the Protein Preparation Workflow.

  1. Includethe entry is represented in the Workspace, the circle in the In column is blue. the homology-out group structure in the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed.
  2. Launch the Protein Preparation Workflow panel from Tasks
    • You can also launch the panel from the Favorites Toolbarbuttons 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.
  3. Specify the Protein by selecting use structures from Workspace (included entry)
    • Your entry name will populate,
  4. Ensure Preprocess is toggled on
  5. Ensure Optimize H-bond Assignments is toggled on
  6. Ensure Clean Up is toggled on
    • This launches a minimization job
  7. Rename the job Model_of_Tyk2_based_on_3ZC6_A-1_prepared
  8. Click Run
    • After this batch job finishes, a new structure is added to the Entry Lista simplified view of the Project Table that allows you to perform basic operations such as selection and inclusion.

Optional: To explore the default settings used for each of these jobs, select More Options and Settings

The Protein Preparation Workflow is an automatic batch job that runs each of the toggled options rather than running each step individually. To learn more, visit the Introduction to Structure Preparation and Visualization tutorial and corresponding documentation page for the Protein Preparation Workflow.

5. Running IFD-MD Job

Now that we have a prepared homology model with the template ligand, we will use IFD-MD to place the target ligand. All of the side chains in the binding site are re-optimized at this step to determine their optimal contacts with the TYK2 ligand of interest.

Figure 5-1. Launching IFD-MD panel.

  1. From Tasks, search for ifd
    • Two items are displayed in the results
  2. Select IFD-MD
    • The IFD-MD panel opens

Figure 5-2. Preparing IFD-MD job.

  1. Include Model of Tyk2 based on 3ZC6_A-1 - prepared complex and select the TYK2 Ligand
  2. Set Target ligand from Project Table and click Load
    • This loads the TYK2 Ligand entry
  3. Click Load for the Template complex in the Workspace
    • Template complex is the Model of Tyk2 based on 3ZC6_A-1 - prepared structure 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.
    • Template ligand(s) is the ligand docked in the Model of Tyk2 based on 3ZC6_A-1 - minimized complex. This will likely be automatically detected. Pick to choose the ligand if it there is more than one ligand in the Entry
    • IFD-MD help
  4. Rename the Job name to be IFD-MD_hm_tyk2

To know 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 Entry List (and Project Table) and the row for the entry is highlighted. Project operations are performed on all selected entries. check the glossary at the end of this tutorial.

Figure 5-3. Adjusting IFD-MD job settings.

  1. Click cog button in the panel
    • Job settings panel opens
  2. Choose a CPU subhost
  3. Indicate the number of processors for the CPU subhost to use (8 CPUs were used in this tutorial)
  4. Choose a GPU subhost
  5. Indicate the number of GPUs for the GPU subhost to use (50 GPUs were used in this tutorial)
    • The panel runs one GPU per subjob
  6. Click Run
    • As the job takes many hours, we will look at pregenerated results with the Prepped 4GIH Aligned on 3ZC6 structure from the Entry Lista simplified view of the Project Table that allows you to perform basic operations such as selection and inclusion..

Figure 5-4. Comparing poses.

To examine the IFD-MD job:

  1. Include Prepped 4GIH Aligned on 3ZC6 from the Entry Lista simplified view of the Project Table that allows you to perform basic operations such as selection and inclusion.
  2. In the Entry Lista simplified view of the Project Table that allows you to perform basic operations such as selection and inclusion., double-click the In circle for Prepped 4GIH Aligned on 3ZC6
    • The entry is fixed in the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed.
  3. Also include the first entry of the IFD-MD_hm_tyk2-out group from the Entry Lista simplified view of the Project Table that allows you to perform basic operations such as selection and inclusion.
  4. Type L on  your keyboard
    • The Workspace structures are zoomed in to the ligands
  5. Compare the two poses of both ligand and protein

6. Setting up FEP+ to Validate the Aligned Poses

FEP+ can be used to evaluate different homology models if there is a set of congeneric ligands with known affinities for the protein being modeled. IFD-MD generates poses that are then used as input for FEP+. The IFD-MD combined score is used to prioritize structures to be used with FEP+.

FEP+ can provide accurate predictions of binding affinity, assuming the IFD-MD structure has a low RMSD from the crystal structure of the structure with the congeneric ligand.

For evaluation of binding site poses with FEP+ it is often useful to perform FEP+ calculations with enough poses to see examples where the retrospective affinity is both accurate and inaccurately reproduced. This provides additional confidence that  the retrospective ligand affinity set contains molecules with sufficient range of affinity and diversity of protein-ligand interactions to differentiate the poses.

Please note that this FEP+ job was completed with the 2021-2 Maestro Release, so your results and panels may vary slightly from those shown below.

6.1 Aligning Ligands

Figure 6-1. Duplicating entry.

  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 Entry List (and Project Table) and the row for the entry is highlighted. Project operations are performed on all selected entries. the first entry of the IFD-MD_hm_tyk2-out group
  2. Right-click the entry and choose Duplicate > Into New Group
    • Duplicate intro New Group panel opens

Figure 6-2. Placing duplicated entry.

  1. Change the New group title to FEP+ validation
  2. Check At top level and choose End of table
  3. Click Duplicate
    • A new entry at the end of the Entry List table is created with the title indicated

Figure 6-3. Splitting an entry.

  1. Click on the FEP+ validation group to 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 Entry List (and Project Table) and the row for the entry is highlighted. Project operations are performed on all selected entries. the entries
  2. Right-click on the group entry and choose Split > Into Ligands, Water, Other
    • A new group inside the FEP+ validation group is created with each splitted structure

Figure 6-4. Selecting multiple entries.

  1. From the tutorial files, import unaligned_ligands_for_fep_validation.mae
  2. Hold the Ctrl key (Cmd for Mac) and select TYK2 Ligand-ligand from TYK2 Ligand_split_by_structure group, and click Unaligned Ligands for FEP Validation group to 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 Entry List (and Project Table) and the row for the entry is highlighted. Project operations are performed on all selected entries. all its structures

Figure 6-5. Ligand alignment settings.

  1. From Tasks launch Ligand Alignment panel
  2. Select Project Table (17 selected entries) from Align ligands from
  3. Select Similar for Ligand structures are
  4. Ensure TYK2 Ligand-ligand is set as the Reference ligand
  5. Check Define common scaffold
  6. Ensure Largest common Bemis-Murcko scaffold is chosen from the list
  7. Click Start at the end of the Ligand Alignment panel
    • A new group with the aligned ligands is added at the end of the Entry Lista simplified view of the Project Table that allows you to perform basic operations such as selection and inclusion.

Figure 6-6. Including all aligned ligands.

  1. Right-click on the newly created group from previous alignment job and select Include
    • A warning message appears asking to continue including more than 10 entries. Click Continue
    • The ligands are all 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 are aligned

6.2 Running FEP+

Figure 6-7. Selecting multiple entries.

  1. Right click on the first entry TYK2 Ligand_ligand and click Delete
    • A warning message appears to confirm deleting the selected entry from the project. Click OK.
  2. Right click on the aligned ligand group and select Exclude
  3. Select TYK2 Ligand_protein from FEP+ validation group
  4. Hold Ctrl key (Cmd for Mac) and select the aligned ligand group

Figure 6-8. FEP+ panel settings.

  1. Launch FEP+ panel from Tasks
  2. In Import structures or perturbation map from, choose Project Table (17 selected entries)
    • If a message appears asking to uniquely rename entries, click OK

Note: There will be issues associated with the receptor because it is a homology model. Ignore these for now.

  1. Click Next

Figure 6-9. Generating map.

  1. Switch to Map tab
  2. Click Generate Map…
    • Map Options panel launches
  3. Keep Map Options panel settings to default with Map topology type to be Optimal
  4. Click Generate Map
    • Wait a few seconds for the map to be generated

Figure 6-10. FEP+ job settings.

  1. Click the cog icon beside Run to launch the Job Settings panel
  2. Change the job Name to FEP_validation_tyk2_homology_model
  3. Change the CPU and GPU host as appropriate
  4. Change the Total GPUs used
  5. Click Run
    • This job takes approximately 8 hours to complete. You will use pre generated results in the next section
  6. Repeat section 6 on the second IFD-MD job output entry as a negative control

7. Analyzing FEP+ Job Results (Optional)

Figure 7-1. Importing pregenerated FEP+ results.

  1. Back in the FEP+ panel, in Import structures or perturbation map from, choose File
  2. Click Browse
  3. Navigate to the tutorial files. Select FEP_validation_for_homology_model folder then choose FEP_validation_for_homology_model_out.fmp
  4. Click Open
  5. Click Next

Figure 7-2. Plotting to compare predicted to experimental affinites.

  1. Click Plot
    • The Correlation Plot panel opens

For best practices, it is recommended to evaluate with FEP+ the top two IFD-MD poses where the second pose will typically be different from the top ranked pose. This second pose can serve as a negative control expected to perform poorly in FEP+, which further validates that you are getting the right answer for the right reasons.

In the following figures, we provide the FEP+ plots for the top two IFD-MD poses. As you can see, the second ranked pose performs poorly with FEP+ showing an R2 of 0.01 compared to the top ranked pose that shows an R2 of 0.89 where the excellent prediction means this homology model is most likely correct, at least for the area of the ligand covered by the SAR data. This FEP job was run with the 2020-2 release of our software, if you are completing this tutorial with a newer release your results may vary slightly.

Figure 7-3a. FEP+ plot for the top IFD-MD pose.

Figure 7-3b. FEP+ plot for the second IFD-MD pose.

If the second IFD-MD pose was very different and yet also gave a good R2 and RMSE in FEP+, then you would conclude that the congeneric ligand series is not broad enough to effectively validate the model.

8. Conclusion and References

In this tutorial we learned how to create a homology model of TYK2, including a bound ligand, using the structure of JAK3 as a starting point. We then compared this homology model with the crystal structure for TYK2 bound to 4GIH ligand. Finally we used FEP+ to analyze the binding site and predict corresponding binding affinities.

For further reading:

 

 

9. Glossary of Terms

Entry List - 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 Entry List (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.