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GLIDE

Structure-Based Virtual Screening Using Glide2016-3

Schrodinger Academy Maestro 11 Tutorials

Structure-Based Virtual Screening UsingGlideThis tutorial demonstrates how to perform a virtual screen for potential inhibitors of FXa using theligand docking application Glide. You will learn how to generate a protein receptor grid, dock a setof ligands into the receptor grid, and analyze the docking results.

Words found in the Glossary of Terms are written in blue, file names are in green, and items that areclicked or typed are bolded.

Createdwith: Release 16-3Prerequisites: Introduction to Structure Preparation and Visualization (Release 16-2 or higher)Filessupplied: 1fjs_prep_complex.maegz; 1fjs_prep_recep.mae.gz; 1jfs_prep_lig.mae.gz;50ligs_epik.mae.gz; factorXa_xp_refine_pv.maegz

This tutorial comprises the following seven parts:

1. Virtual Screening Prerequisites - Protein Preparation Wizard, LigPrep2. Creating Projects and Importing Structures - Maestro3. Generating a Receptor Grid - Glide4. Docking the Cognate Ligand and Screening Compounds - Glide5. Analyzing Results and Binding-Site Characterization - Pose-Viewer, XP Visualizer, SiteMap6. Conclusion and References7. Glossary of Terms

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1. Virtual Screening PrerequisitesStructure files obtained from the PDB, vendors, and other sources often lack necessary informationfor 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 modelingtasks, we use the Protein Preparation Wizard to resolve issues.

Similarly, ligand files can be sourced from numerous places, such as vendors or databases, often inthe form of 1D or 2D structures with unstandardized chemistry. LigPrep can convert ligand files to3D structures, with the chemistry properly standardized and extrapolated, ready for use in virtualscreening.

In this tutorial, the protein, cognate ligand, and virtual screening ligands have already beenprepared in order to save time. However, these preparation steps are a necessary part of a virtualscreen and must be done before docking. Please see the "Introduction to Structure Preparationand Visualization" tutorial for instructions on using the Protein Preparation Wizard and LigPrep.

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2. Creating Projects and ImportingStructuresProjects (*.prj extension) are the main file format for Maestro. A project file may contain numerousentries corresponding to imported structures, as well as the output of modeling-related tasks. Oncea project is created, the project file is automatically saved each time a change is made.

2.1 Create a project in the Working Directory

At the start of each session, change the file path to your chosen working directory in Maestro. This makesfile navigation easier. Every session in Maestro begins with a default Scratch Project. Scratch Projects aretemporary and not saved; you will have to name the Scratch Project in order to save all subsequent work.

Double-click the Maestro icon

Figure 2-1. Change Working Directoryoption and dialog.

Navigate to File > Change Working Directory…,select your directory and click Choose...

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Figure 2-2. Save Project dialog.

File > Save Project AsChange the File name to “FXa_glide”, click Save

The project is now named FXa_glide.prj

2.2 Set up the pre-installed files

Figure 2-3. The Tutorials panel, showingfiltered results with “Glide” as a keyword.

Pre-installed files for this tutorial can be copied over from theinstallation directory. Copy the files for the Glide Quick StartGuide into your working directory:

Help > Tutorials… and select the Glide Quick StartGuideNote: In Filter, type "Glide" to find the guide easily.Click  Copy...

All tutorial files are copied to your WorkingDirectory

Click  Close

Figure 2-4. The import panel, with non-contiguous files selected

With the files copied into your Working Directory, we will nowimport the necessary subset into Maestro for this tutorial.

Note: *.mae  and *.mae.gz files are the default structure fileformats for Maestro. However, all common structure file typesare supported.

File > Import Structures…Select files 1fjs_prep_lig.mae.gz,1fjs_prep_recep.mae.gz, and 50ligs_epik.mae.gzfrom your working directory. Use Ctrl-click (or Cmd-click on a Mac) to select multiple, non-contiguous files.Click  Open

Structures are now shown in the Entry ListA banner will appear confirming four entries havebeen imported

Note: The imported structures are automatically added to theEntry List (located to the left of the Workspace) and ProjectTable (Ctrl+T or Cmd+T). The Entry List is located to the left ofthe Workspace.

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3.1 Identify the Binding Site

3. Generating a Receptor GridGrid generation must be performed prior to running a virtual screen with Glide. The shape andproperties of the receptor are represented on a grid by fields that become progressively morediscriminating during the docking process. To add more information to a receptor grid, differentkinds of constraints can be applied during the grid generation stage. For a comprehensive overviewof constraint options, see the grid generation videos on our website or the Glide User Manual("Help" > Help... > User Manuals > Glide User Manual). In this tutorial, we will set a hydrogenbond constraint.

Figure 3-1. The Receptor Grid Generationdialog.

Figure 3-2. Identifying the binding siteregion. The ligand is picked (greenhighlights) so that it can be excluded fromthe grid generation calculation.

Click "In" circle to include 1fjs_prep_complex in theWorkspaceNavigate to Tasks > Browse > Docking and VirtualScreening > Receptor Grid Generation circle toinclude 1fjs_prep_complex

The Receptor Grid Generation dialog opens

Note: Receptor Grid Generation is part of the Favorites Bar bydefault. It can also be accessed by the searching function inTasks.

In the “Receptor” tab under Define Receptor, check theboxes for Pick to Identify the ligand (Molecule) andShow Markers

A banner in the Workspace will prompt you toclick on an atom in the ligand. Predictivehighlighting can assist with choosing a ligandatom.

Click on the ligand in the Workspace.The ligand is now highlighted in green with apurple box around it. The box defines theoutermost region that the docked molecule(s)must occupy in order to pass the initial stages ofdocking.

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3.2 Define the Bounding Box Dimensions

3.3 Set a Hydrogen Bonding Constraint

Figure 3-3. Ligand diameter midpoint boxdialog and adjusted green bounding box

Click on the "Site" tabThe Centroid of the Workspace ligand (selectedin the Receptor tab) option is chosen.

Note: In this case, the position of the purple bounding box isdefined by the co-crystallized ligand, but other options areavailable. For example, if you are working with an apo-structure, the binding site region can be defined by pickingresidues.

Click on Advanced Settings...A green inner bounding box appears. Thisdefines the region in which the centroid of thedocked molecule(s) must occupy in order to passthe initial stages docking.

Adjust the settings for X, Y, and Z sizes to 10, 8, and 6Å, respectively.

The shape of the green box is changed.

Note: This feature is useful when the virtual screening ligandsare a different size to the cognate ligand. The size of theboxes define the screening volume, which influences theruntime of the job. The larger the box, the longer the runtime.

Click OK

Figure 3-4. Hydrogen bonds are shownbetween the ligand and ASP 189.

Type L to zoom into the ligand and find the hydrogensthat form H-bonds to ASP 189

Note: Please see the Introduction to Structure Preparationand Visualization tutorial for instructions on how to addresidue labels and show H-bonds.

Click on the "Constraints" tab and choose H-bond/Metal"

A banner will prompt you to choose an atom inthe receptor

Click on the oxygen atoms of the ASP 189 residue.Crosshatches appear over these atoms in theWorkspace and a new constraint gets added inthe panel. By default this carbonyl constraint willbe treated as symmetrically equivalent by Glide.

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3.4 Generating the Grid

Figure 3-5. The Receptor Grid Generationdialog showing the H-bond constraint forASP 189.

At the bottom of the Receptor Grid Generation dialog,change the job name to 1fjs_gridClick RunCheck the Job Monitor to determine the progress - thejob typically takes a few minutes.

A grid file named 1fjs_grid.zip will be generatedin your working directory

Close the Receptor Grid Generation dialog

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4. Docking the Cognate Ligand andScreening CompoundsThe minimum requirements for running a virtual screen in Glide are a grid file and a ligand file.Optionally, you can set ligand- and receptor-based constraints, and incorporate chemical similarityinto the final docking score. For a full explanation of these topics, consult the Glide User Manual.Here we will not set any additional constraints, but we will include the hydrogen bonding constraintthat we created in the previous step.

Prior to docking the screening compounds, we will run a preliminary step of re-docking the cognateligand. Cognate ligand docking is a way to establish a benchmark for a virtual screen ofcompounds with unknown binding activity against a target. The information gained from this stepcan help with evaluating poses and beneficial interactions, which is useful for lead generation.

The steps below describe how to dock the cognate ligand file, 1fjs_prep_lig.mae.gz, and thescreening compounds file, 50ligs_epik.mae.gz, in a separate run. Both jobs require docking to thesame receptor grid file that was generated in the previous step.

4.1 Dock the cognate ligand file

Figure 4-1. Ligand docking dialog.

Navigate to Tasks > Browse > Docking and VirtualScreening > Ligand Docking...Note: This can be added to your Favorites Bar byclicking the star to the left of Ligand Docking.

Next to Receptor grid window, click Browse... andchoose 1fjs_grid.zip in your working directoryNext to the Use ligands from window, choose FilesIn the File name window, click Browse... and choose1fjs_prep_lig.mae.gzChange the Job name to 1fjs_glide_cognate and clickRun

The job will take ~1 minute to complete and fileswill be incorporated into the Maestro Entry Listand Project Table.A banner appears in the Workspace when the filehas incorporated

Include the output 1fjs_prep_lig file in the Workspaceand select the whole ligandRight-click on the ligand and choose Style to make theligand greenNote: See Introduction to Structure Preparation andVisualization for further details on styling in theWorkspace

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4.2 Dock the screening compounds

Figure 4-2. The cognate ligand (grey)bound in 1FJS with the SP-docked Glidepose (green) in good agreement.

Include the 1fjs_prep_complex structure with1fjs_prep_lig still in the Workspace

Figure 4-3. Ligand Docking dialog set upfor screening compounds.

In the Entry List, select the group 50ligs_epikIn the Ligand Docking dialog, change the Use ligandsfrom window to Project Table (Selected Entries)Note: Keep 1fjs_grid.zip as the receptor grid

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Figure 4-4. The “Constraints” tab showingone H-bond constraint will be used.

Click the "Constraints" tabClick the Use box to include the hydrogen bondconstraint in the docking job

Figure 4-5. Ligand Docking Outputsettings.

Click the "Output" tabClick the box to Write per-residue interaction scoresChange the Job name to 1fjs_glide and click Run

The job will take several minutes to complete andfiles will be incorporated into the Maestro EntryList and Project TableA banner appears in the Workspace when the filehas incorporated

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5. Analyzing Results and Binding-SiteCharacterization5.1 Visualize the results through Pose Viewer

Figure 5-1. Visualizing a screeningcompound using the Pose Viewer

Multiple Glide job results can be in the Entry List and beidentified by the job name. Docked results will show thereceptor in the first row and the docked ligand(s) in thesubsequent row(s), where they are ordered by best to worstGlide Score, or Docking Score if ‘Epik’ was used in setting upthe job. The Glide Score is broken down by van der Waalselectrostatic components and can be seen more easily in theProject Table using the Property Tree.

Navigate to Tasks > Browse > Docking and VirtualScreening > Pose Viewer...Click Import File... and choose 1fjs_glide_pv.maegzfrom your Working DirectoryClick Set Up and check the box to display Per ResidueInteractionsToggle through the results using the right and leftarrow keys.

Ligand poses are displayed in the WorkspaceResidues are colored according to theirinteraction energies, ranging from red(unfavorable) to blue (favorable)

Note: Interactions can be adjusted using the Pose Viewer orthe Interactions button in the Workspace Configurationtoolbar

5.2 Analyze the results

Figure 5-2. Primary Glide properties areshown in the Project Table.

The Project Table contains the Glide Score along with thecomponents of the score calculation. Use the property tree tosee specific columns of energies, which have been split intothe most relevant properties (primary) and less relevantproperties (secondary).

In the Project Table, click the property Tree iconThe property tree appears to the right of theProject Table

Click the All box, twice to deselect all boxesClick on the Glide box and select Primary

Only the docking score, glide gscore, and glideemodel columns are shown in the Project Table

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5.3 Visualize pre-docked XP results

Figure 5-3. The XP Visualizer showingrewards (blue) and penalties (red) to theGlide Score.

In this tutorial, Glide XP, with write XP descriptor information,has already been performed using a subset of the screeningcompounds. For more details on running Glide XP, see theGlide User Manual ("Help" > Help…> User Manuals > GlideUser Manual). This has been done to show the individualcomponents of the scoring function and demonstrate howvarious rewards and penalties associated with dockingcontribute to the Glide Score. Results can be visualized in atable or in the Workspace.

Navigate to Tasks > Browse > Docking and VirtualScreening > Visualize XP Interactions...Click Open... and selectfactorXa_xp_refine_pv.maegz from your WorkingDirectoryChoose Docking Score as the activity property, clickOK

The tab is populated with XP resultsIndividual terms of the scoring function arecolored as red (penalties) or blue (rewards)

Click on the colored entries to visualize in theWorkspaceClick Export Data... to export the spreadsheet as aCSV file

5.4 Identify a binding site with SiteMap

Figure 5-4. SiteMap dialog

SiteMap characterizes hydrophilic, hydrophobic, acceptor anddonor regions of a receptor by way of a volume map. This isuseful to learn more about an active site, predict a binding siteif it is unknown, or if allosteric sites are of interest. SiteMapcan calculate possible binding regions over the protein andranks the sites by way of a druggability score. The outputfrom a Glide virtual screen can be overlaid with SiteMapinformation to examine how well the docked ligands explorethe various regions in the binding cavity. This is a helpful stepfor lead optimization.

Include 1fjs_prep_complex in the Workspace.Navigate to Tasks > Browse > Structure Analysis >Binding Site DetectionIn the Task section, choose Evaluate a Single BindingSite RegionClick on the ligand in the Workspace

The ligand is highlighted in green

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Figure 5-5. The cognate ligand is shownwith the SiteMap for 1FJS

This allows SiteMap to remove the ligand duringthe calculation

Change the Job name to sitemap_1fjsClick Run

A banner appears when the job has incorporatedInclude all three results in the Workspace, zoom intothe ligand by typing L

Various surfaces are shown representing differentregions of hydrophilic property; hydrophobic(yellow), acceptor (red), donor (blue).The original site points (white spheres) eachrepresent ~1 Å

Click on the S next to sitemap_1fjs_site1 in the EntryList to modify the surfaces

Note: To find other possible binding sites on 1FJS usingSiteMap, chose “Identify top-ranked potential receptorbinding sites” in the Task section.

3

5.5 Generate a Receptor Grid from SiteMap

Figure 5-6. SiteMap points have been usedto generate a receptor grid.

Sites identified by SiteMap can be used to create receptorgrids for virtual screening experiments. This can be useful forexploring sites without a known active compound.

Include 1fjs_sitemap_find_site_1 and1fjs_sitemap_find_protein in the WorkspaceNavigate to Tasks > Browse > Docking and VirtualScreening > Receptor Grid GenerationSelect Pick to identify ligand, and choose Entry fromthe option menuClick on a site point near the center of the hydrophobicsurface region (yellow)Change the Job name to 1fjs_sitemap_entryClick Run

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6. Conclusion and ReferencesIn this tutorial, we completed a workflow for virtual screening using Glide. Building off lessons learned fromIntroduction to Structure Preparation and Visualization tutorial, we used cognate ligand docking tobenchmark our virtual screen. Then, a series of screening compounds were docked and the results wereviewed using Pose Viewer, with known actives being the top hits. SiteMap was used to explore the bindingsite and generate another receptor grid. The information gained from this virtual screen can be used to findligand candidates for further Structure-Based Lead Optimization with Glide & MM-GBSA.

Quick Start and User Manuals

Video Guides

Other tutorials

Maestro Quick Learn Tasks

Structure-Based Virtual Screening Using Glide 14 of 15

6. Glossary of Termscognate ligand - a ligand that is bound to its protein target

Entry List - a simplified view of the Project Table that allows you to perform basic operations such asselection and inclusion

included - the entry represented in the Workspace, the circle in the “Included” column is blue

incorporated - once a job is finished, output files are then copied back to the working directory

Project Table - displays the contents of a project and is also an interface for performing operations onselected entries, viewing properties, and organizing structures/data

Scratch Project - a temporary project in which work is not saved, closing a scratch project removes allcurrent work and begins a new scratch project

selected - the entry is chosen in the Entry List, the row is blue, operations will be

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

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