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Tutorial: Exploring Protein-ligand BindingJune 3, 2014

CLC bio, a QIAGEN CompanySilkeborgvej 2 Prismet 8000 Aarhus C DenmarkTelephone: +45 70 22 32 44 Fax: +45 86 20 12 22www.clcbio.com [email protected]


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Tutorial: Exploring Protein-ligand Binding


This tutorial takes you through how protein-ligand binding can be explored using CLC Drug

Discovery Workbench. The development of inhibitors of cyclin-dependent kinase 2 (CDK2) is usedas an example.

Typically, a drug target is a specific key protein involved in a particular metabolic or signalingpathway specific to a disease condition. For a drug (ligand) to hit the target with high affinity andspecificity, it should be complementary in shape and electrostatics to the active region of thetarget protein. 3D modeling of the protein-ligand interaction obtained via docking simulations istherefore an invaluable tool in the process of drug design. It helps you to

Understand the mechanism of ligand binding to the target.

Understand why one compound binds more strongly than another. Spawn design ideas for improved binders.

Test design ideas before proceeding to synthesis.

Example data relating to this tutorial can be imported from the Helpmenu (Help | Import ExampleData). The files for this tutorial are found in CLC_Data/Example Data/Protein-ligand docking.

You can work quickly through the tutorial, just doing the actions listed with blue background.The header titles through the tutorial match the titles of the blue boxes in figure1.

Import Protein Structure

Molecular docking is performed against a target protein structure. The Protein Data Bank holdsmore than 90 thousand protein structures. To find the most relevant structure to use, you cango to www.pdb.org and search for it, or use the built in Search for PDB Structures at NCBI optionfrom the Download menu.

For this tutorial you should download the PDB file with ID 1KE5 (see figure2).

Go to the Download menu and select Search for PDB Structures at NCBI...Write the PDB ID 1KE5 in the search field and press Start search.

Double click on the row that appears.

The molecule structures listed in the PDB file will then open in a Molecule Project, showing thecontent in a 3D view (see figure3).

Save the Molecule Project, by clicking the Save button in the Toolbar

or select Save in the File menu. A Molecule Projectfile named 1KE5 appears in the NavigationArea.

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Figure 1:Schematic of the tutorial workflow. The Molecule Project is central in the work. It containsmolecule structures that can be visualized in 3D. Molecule Tables and Docking Results Tablescontain rows of molecules with 2D depictions. Example file names are shown in italic.

The protein target and other molecules imported with the protein can be visualized using theProject Tree and visualization options available in the Side Panel. Sequence alignment andanalysis tools can also be used to find out more about the protein. This is described in theExplore your Proteintutorial.

Find Binding Pockets

A molecular docking is aimed at a specific region of the target, expected to be the binding site.You may know the location of this site based on the position of ligands or cofactors co-crystallizedwith the protein structure, or positions of amino acids known to participate in the binding. If youdo not know the binding site, you can use the Find Binding Pockets tool found in the Toolbox.

Invoke the Find Binding Pockets tool found in the Drug Design folder in the Toolbox.

Step 1: Select the Molecule Project 1KE5 as input.

Step 2: Leave the parameters at their defaults setting.Step 3: Choose toOpenthe results and click Finish.

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Figure 2:The built-in Search for PDB Structures at NCBI option.

Figure 3: The content of the PDB file 1KE5 imported to a Molecule Project (this visualization issaved as view 'Just downloaded' on the example file 1KE5).

Info box: Druggable binding pockets

Analysis of 5600 protein-ligand structures from the PDB has revealed that 95 % of bindingsites are within one of the three largest solvent-accessible pockets found on the protein[Liet al., 2008]. In the Find Binding Pockets tool, the lower limit for pocket volumes canbe specified. Pockets with good drug-binding properties are typically, but not always,compact [Cheng et al., 2007]. The tool will also look for more exposed pockets, if 'Includemore exposed pockets' is checked (see figure4).

Now some binding pockets will appear in the Project Tree.

Use the check boxes in the Project Tree to display the found binding pockets one by one.

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Figure 4:Parameter settings for the Find Binding Pockets tool.

In this case we already knew where to locate the binding site, as the protein structure came witha co-crystallized inhibitor. As expected, we see that the largest pocket returned from the FindBinding Pocket tool overlaps with the inhibitor (LS1) position as seen in figure 5.

Figure 5: Binding pocket indicated with green spheres. The co-crystallized ligand seen in ball-and-sticks representation (this visualization is saved as view 'Largest pocket' on the example file '1KE5pockets found').

Setup Binding Site

Click the Setup Binding Site button found below the Project Tree in the Side Panel.

This will raise the Setup Binding Sitedialog box as seen in figure6.

The binding site volume to target with the dockings should be specified first. The center of thesite can be based on co-crystallized ligands, binding pockets found using theFind Binding Pocketstool, or on a set of atoms selected in the 3D view. In this example, we have a co-crystallizedligand, LS1, which we can use as center. It is important to make sure that the binding sitesphere/volume is set large enough, so that all ligands you plan to dock against the protein canfit inside it. For this example, the default radius of 13 is fine.

An automatic binding site setup is performed inside the binding site volume. The setup can be

inspected and manually altered via the dialog box. Be aware that all molecules included in thebinding site setup, i.e. protein chains, cofactors, nucleic acids, and water, are treated as rigidentities during the docking, and the ligands docked to the binding site will not be allowed to

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Figure 6:The Setup Binding Site dialog box with the binding site volume indicated as a transparentgreen sphere.

overlap with these molecules. You can go through the categories in the dialog box, and inspectthe settings and e.g. try changing the protonation of one of the amino acids.

Clicking the Help button in the bottom of the dialog box, will take you to a detailed description ofthe Binding Site Setup dialog box in the manual.

Info box: Binding Pockets

The same algorithm is used to find binding pockets in the Find Binding Pockets tool andin the Setup Binding Site dialog box. In the case of the Find Binding Pockets tool, pocketsare found on the union of all protein chains in the Molecule Project. In the case of the

Setup Binding Site dialog box, pockets are found on the union of all molecules includedin the binding site setup.

For this example, the automatic binding site setup should be fine, so no changes to the setupare needed.

Press the OK button.

YourMolecule Projectnow has aBinding Site Setupincluded, which appears in the Project Tree,

and your protein target is now ready for docking. You can always inspect and change the BindingSite Setup of theMolecule Projectby re-invoking the Setup Binding Site dialog box.

Info box: Sharing a Binding Site Setup

If you have a CLC Drug Discovery Server, the Molecule Project holding the binding sitesetup should be placed on the server, such that other researchers working on the drugdiscovery project can have easy access to make their own copy of the Molecule Projectfor further exploration and individual docking studies. Alternatively, the Molecule Projectcan be exported as a CLC object and distributed.

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Validate Setup

Inspect Co-crystallized Ligand

When a protein structure is downloaded from the Protein Data Bank, it will often come with a co-crystallized ligand. This ligand appears in the Ligands category in the Project Tree after download.It is always a good idea to begin your docking studies with a docking of the co-crystallized ligand,to check if the docking simulation can recreate the correct binding mode for this known binder.

To model protein-ligand binding, it is important that the representation of the molecules isphysically and chemically sound. Preparing a molecule for docking thus means taking carethat it is represented with the proper connections between atoms, bond orders, hydrogen atompositions, and atom hybridizations. As much of this information as possible is taken from theinput (in this case the PDB file). The rest is automatically assigned by the workbench based onthe given input. Using the ball'n'sticks visualization ( ) for the ligand molecule, the assignmentof bond connections, bond orders, and hydrogen atom positions are clearly visualized in the 3Dview (see figure7). The assigned atom hybridizations can be inspected from the Property viewerin the Side Panel, when holding the mouse over an atom.

Figure 7: The co-crystallized ligand showing the representation automatically assigned based onthe PDB input.

In the Issues list ( ), potential issues with the representation of the molecules in theMoleculeProjectare listed (figure8).

Right-click in an empty space in the 3D view and select Show | Issues.

Sort the issues after molecule by clicking the header of the Molecule column.

Selecting issues in the list will zoom to and highlight the involved atoms in the 3D view. Noissues are found related to the ligand on import, but in the next section, corrections will be madeto the ligand representation, that will make issues appear in the list.

Correct the Ligand Representation

If there is something you wish to change about the representation of a molecule, you canmanually adjust the atom and bond properties via the context menu on the atom in focus in the3D view. In this case, the chemical group -N=CH2does not represent the molecule added to the

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Figure 8:The Issues list of the Molecule Project seen in split-view with the co-crystallized inhibitor.

protein. In the paper describing the protein structure[Bramson et al., 2001], the chemical groupof the inhibitor is described as -NH-CH3. The reason why the workbench has assigned a doublebond in this location is the S-N-C angle, which is very different from what would be expected fora group with single bonds. You can measure this angle by clicking the three atoms S-N-C whileholding down Ctrl (Cmd on Mac), then the angle will be shown in the Property viewer found in theSide Panel (129 degrees).

You should now change the representation of the chemical group. When changing bonds for anatom, the neighboring atoms are listed with their atom names, such as N33 for a nitrogen. Theatom names can be displayed by selecting the ligand in the Project Tree and clicking the Labelbutton below the Project Tree.

Move the mouse pointer to the terminal carbon atom and right-click.

Set hybridization | SP3

Set hydrogen count | 3

Set bond order for | Bond to N33 | Single

Then move the mouse pointer to the nitrogen and right-click.

Set hybridization | SP3

Set hydrogen count | 1

While making the changes to the representation, you should see issues appear and disappearin the Issues list - as seen in figure9. In the end you should have no issues left related to theligand molecule.

Even when there are no issues left on the molecule, you should make sure the representationadheres to your knowledge about the molecule.

Save the Molecule Project.

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Figure 9:Chemistry issue appearing in the Issue list while changing molecule representation.

Now the ligand is ready to be docked to the binding site.

Fast Track Docking

The molecules found in the Ligands category in the Project Tree can be docked to the BindingSite Setup of theMolecule Project, if such is present.

Select the co-crystallized ligand by clicking on it in the Project Tree.

Click the 'Dock Ligand' button found below the Project Tree (see figure 10).

The docking simulation will now start in the background. When the docking is done, the bestscoring binding mode, found by the docking simulation, will appear in the Docking results categoryin the Project Tree.

The docking simulation is stochastic, and you will not get exactly the same binding mode returnedfor each time it is run, even with the same input.

Use the checkboxes in the Project Tree to display the Docking resultLS1 together with theco-crystallized ligand LS1, to compare their binding modes as exemplified in figure 11.

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Figure 10:Select one or more ligands in the Project Tree and press Dock Ligand.

Figure 11: Comparing the binding mode of the co-crystallized ligand with the docking result (thisvisualization is saved as view 'Binding modes compared' on the example file '1KE5 setup validated').You can right-click quick-style buttons (or click-hold) to get menus with available color schemes.

Figure11shows the co-crystallized ligand in ball-and-sticks representation and the binding modereturned from the docking of the ligand in sticks representation with brown carbon atoms. Thecore of the ligand, where the important protein interactions are found, are accurately establishedin the docking result.

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Info box: Failure to re-establish known binding mode

If a co-crystallized ligand does not establish its known binding mode in the docking, thesetup of the binding site and the preparation of the ligand should be re-evaluated. Also,the co-crystallized ligand can be extracted from the Molecule Project using the ExtractLigands tool in the Toolbox. This will put the ligand in a Molecule Table that can be usedas input for the Dock Ligands tool found in the Toolbox and described in the following.That will give access to tune parameters related to the sampling of binding modes.

Inspect Docking Result

To study how the protein interacts with the ligand, select either the ligand LS1 or the dockingresult LS1 from the Project Tree, and invoke the right-click context menu. Pick Binding Site

Interactions | Show Hydrogen Bondsto display protein residues forming hydrogen bonds to theligand, with the hydrogen bonds shown as blue dashed lines. UseBinding Site Interactions | HideHydrogen Bondsto hide them again.

Pick Binding Site Interactions | Create Interacting Atoms Group to generate an atom groupconsisting of protein residues, and molecules included in the binding site setup, which have atleast one heavy atom within 5 of a ligand heavy atom. The atom group appears in the Atom

groupscategory in the Project Tree. It can be un-displayed and the visualization changed usingthe quick-style buttons found in the bottom of the Project Tree, just like for molecule entries.

Individual residues of particular interest can also be selected from the atom group or fromthe hydrogen bond visualization. Double-click on an atom to select all atoms in that residue.

Invoke the context-menu on the Currentselection in the Project Tree, and pick Create group fromselection. Then a new atom group will appear, only containing the selected residue, and the atomgroup showing all residues around the ligand can be hidden.

Import Small Molecules

We can now go on to import other molecules that we wish to see binding to the target protein.Molecules you wish to see binding to the binding site can be pasted directly into the MoleculeProjectas SMILES strings, which is handy if you have sketched the molecule in a 2D sketchingprogram (see theDock Ligands from a 2D Molecule Sketchtutorial). You can also import moleculeswith 3D coordinates to the workbench as a Molecule Tableor add them to the Molecule Project

holding the binding site setup. In both cases, the file formats SDF, Mol2 and PDB are supported.If the molecule is imported using the Add Molecules to Molecule Project... importer, it will appearin the Ligands category in the Project Tree, and can be docked the same way as the co-crystallizedligand. Alternatively, the molecule can be imported to a Molecule Table, and docked to theBinding Site Setup in the Molecule Project using the Dock Ligandstool found in the Toolbox.This is what we will do now.

Using theStandard ImportorImport molecules with 3D coordinates...options, molecule structuressaved in Mol2 or SDF format can be imported to aMolecule Tablefrom your computer file system.For this tutorial, an SDF file with three ligands has been imported (example file:3compounds).

The compounds are taken from the same inhibitor study as the protein crystal structure [Bramson

et al., 2001]. The co-crystallized inhibitor is compound 98 in that study, and the three compoundsin the example file are compound 32, 72, and 90.

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Inspect Molecules

Double-click the file 3compounds in the Example Data in the Navigation Area to open it. Then

right-click on a row and select Show | Issues, to check that there are no issues associated withthe molecules. In this case, no issues are found, and the Issues list can be closed again.

The molecules are visible as 2D depictions in the table view, and each entry holds informationabout atom coordinates in 3D space. To see the molecules in 3D, click the Select View actionin the table Side Panel. Then choose to view the molecules in 'New molecule project'. You canlook at the molecules in 3D, by selecting the rows in the table. Close the 3D view, showing themolecules from the table.

Dock Ligands

Invoke the Dock Ligands tool wizard from the Toolbox.

In Step 1, select the Molecule Table 3compounds, holding the ligands to dock, as input.

In Step 2, select the Molecule Project with the Binding Site Setup as 'Binding site', andleave the other parameters at their default values (figure12). Note, the wizard will alwaysremember the parameters you used last. To return to the default settings, click thearrow-button in the lower left corner.

In Step 3, choose to Open the results, and click Finish.

Figure 12:The default parameters for molecular docking.

The docking will then start, and the process can be followed from the Processes tab in theToolbox panel. The docking simulation will output the results in a Docking Results Tablethat willopen when the docking is done.

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Info box: The Dock Ligands tool versus the Dock Ligand button.

Invoking the Dock Ligands tool or clicking the Dock Ligand button result in the sameoperations, the only difference is how the input is selected, the output presented, and ifparameters can be customized.

Dock Ligands tool in Toolbox:A number of parameters can be customized, e.g. thelevel of sampling. Ligands should be in Molecule Tables, and all molecules in thetables selected as input are docked. Docking results are presented in a DockingResults Table.

Dock Ligand button in Side Panel: No parameters can be customized, the defaultvalues are used. Only ligands in the same Molecule Project as the Binding SiteSetup can be docked. Only selected ligands are docked. Docking results are directly

added to the Molecule Project.

Inspect Docking Results

The docking simulation outputs a 3D model of how each ligand might bind to the protein (thebinding mode), as well as a scoring of this binding (see the Info box: Scoring a Binding Mode).No matter if the docking has been carried out from inside a Molecule Project, or via the DockLigandstool like in this case, both the binding mode and the score are easily accessible.

TheDock Ligandstool outputs aDocking Results Tablewhere 2D sketches of the docked ligands

can be seen, together with information on the scores of the ligand binding modes.

Make sure the Molecule Project holding the Binding Site Setup is open in the View Area.

From the Side Panel of the docking results table, use the Select View action to connectwith the Molecule Project containing the Binding Site Setup.

Make sure the protein is visible in the Molecule Project.

Browse through the table, to see the generated protein-ligand complexes in the 3D view.

Use the options in the table Side Panel to show or hide hydrogen bonds or nearby proteinatoms for the docked ligands.

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Info box: Scoring a Binding Mode

A particular binding mode of a ligand in the protein binding pocket is connected with ascore. The molecular docking simulation searches through numerous potential bindingmodes of the ligand in the pocket, and the one resulting in the best score is returned fromthe docking. The score mimics the potential energy change, when the protein and ligandcome together. This means that a very negative score corresponds to a strong bindingand a less negative or even positive score corresponds to a weak or non-existing binding.

Score = Stargetligand + Sligand

TheStarget-ligandterm is a sum over contributions from all heavy atom contacts between theligand and the molecules included in the binding site setup. It scores the complementaritybetween the binding site and ligand by rewarding and punishing different types of heavy

atom contacts (inter atom distance below ~5.5 ). The target-ligand score is split inthree types of contributions in the Docking Results Table; Hydrogen bond score, Metalinteraction score, and Steric interaction score.

Rewarded contacts Punished contacts

Hydrogen bond interactions Too close contacts (clashes)

Lone-pair - metal ion interactions Hydrogen bond donor-donor contacts

Non-polar interactions Hydrogen bond donor-metal contacts

Hydrogen bond acceptor-acceptor contacts

Contacts between non-polar atoms and hydrogenbond donors and acceptors

The Sligand term punishes internal heavy atom clashes in the ligand and strain resultingfrom unfavorable bond rotations. This contribution to the score is listed in the DockingResults Table as Ligand conformation penalty. As the score is a sum over contributions,a large ligand can get a better score than a small one, simply due to its size. Whencomparing scores for different molecules, this effect has to be considered and kept inmind.

As the search for best binding mode is stochastic, the final binding mode, and thus the score,will not be identical between docking simulations. The score mimics the potential energy changeon ligand binding, but cannot be directly compared to any experimentally measured value. Themain purpose of the binding mode score is to return the most likely binding mode of a particularligand, the second purpose is to compare binding strength between ligands, which is a muchharder task. In the table below is shown the measured inhibitory effect (Kinase IC 50) of the fourcompounds included in this tutorial[Bramson et al., 2001]. Compound 98 is the co-crystallizedligand.

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Compound Kinase IC50(nM) Score90 4.3 -66.95

32 13 -71.0798 560 -57.8272 >10,000 -52.92

The docked ligands appear as guests in the Project Tree of the Molecule Project. If you prefer tokeep one or more of the bound ligand conformations in the Molecule Project, for future referenceand inspiration, molecules selected in the table can simply be copied to the Molecule Projectusing the Copy Selected to Projectaction in the table Side Panel. The selected table entries willthen appear in the Docking results category in the Project Tree, and will be saved together withtheMolecule Project. The score of the result will be shown in the Property Viewer palette in theSide Panel, when the docking result is selected in the Project Tree.

Continue the Exploration

Inspired by the structural model of the ligand binding, you can sketch up new ideas for binders,import them into the workbench and dock these new molecules to the same Binding Site Setup.You can also use the Ligand Optimizer, found below the Project Tree in the Molecule Project, toplay around with ligand modifications in the binding site.

Maybe, it is also worth playing around with the Binding Site Setup, maybe include some watermolecules in the binding site, or try to use another protein structure as target, if available. There isa section in the Drug Design chapter of the manual on Improving docking and screening accuracy,which describes aspects to consider regarding Binding Site Setup, ligand representation, and the

docking simulation itself.You can also make the Molecule Project, including the Binding Site Setup and a selection ofinteresting docking results, available for other researchers on the project, so that they can inspectand potentially continue the protein-ligand docking studies on their own. If you wish to continuethe modeling exploration of the protein-ligand complexes using other software, Molecule TablesandMolecule Projectscan be exported in Mol2 format.

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References

[Bramson et al., 2001] Bramson, H. N., Corona, J., Davis, S. T., Dickerson, S. H., Edelstein, M.,

Frye, S. V., Gampe, R. T., Harris, P. A., Hassell, A., Holmes, W. D., Hunter, R. N., Lackey,K. E., Lovejoy, B., Luzzio, M. J., Montana, V., Rocque, W. J., Rusnak, D., Shewchuk, L., Veal,J. M., Walker, D. H., and Kuyper, L. F. (2001). Oxindole-based inhibitors of cyclin-dependentkinase 2 (cdk2): Design, synthesis, enzymatic activities, and x-ray crystallographic analysis.

Journal of Medicinal Chemistry, 44(25):4339--4358. PMID: 11728181.

[Cheng et al., 2007] Cheng, A. C., Coleman, R. G., Smyth, K. T., Cao, Q., Soulard, P., Caffrey,D. R., Salzberg, A. C., and Huang, E. S. (2007). Structure-based maximal affinity modelpredicts small-molecule druggability. Nat Biotechnol, 25(1):71--75.

[Li et al., 2008] Li, B., Turuvekere, S., Agrawal, M., La, D., Ramani, K., and Kihara, D. (2008).

Characterization of local geometry of protein surfaces with the visibility criterion. Proteins,71(2):670--683.

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