Structural Bioinformatics Practical Session

Today, in the basic part you will learn now to use the automated docking software Autodock3 and AutoDockTools for docking the ligands in the active site of a protein. In the advanced part, you will perform a similarity search with specified properties and molecular constitution based on the natural ligand, dock the hits and rank based on their docking energies. The protein used in the current exercise is trypsin. Trypsin is a proteolytic enzyme found in the small intestine which hydrolyses proteins into smaller peptides or aminoacids. The aspartate residue (ASP189) in the active site is responsible for this activity. Trypsin cleaves the proteins at the Cterminal side of the amino acids lysine and arginine except, when either is followed by proline. Keywords: AutoDock, AutoDockTools, In-silico Docking, Similarity Search and Virtual Screening

Basic Part - In-Silico Docking

The aim of this part is to get used to the docking software by re-docking the ligand present in the crystal structure of a protein using an automated docking suite called 'AutoDock'. The GUI for AutoDock is AutoDockTools (ADT), which will be used to perform the entire docking task. More information is available on the AutoDock suite homepage http://autodock.scripps.edu/ The latest versions of ADT and autodock are available from the home page. Getting Started 1. Open a terminal window. Create a directory called 'docking' by typing 'mkdir docking'.Type 'cd docking' to enter the docking directory.

Important
You need to set the stack size unlimited for AutoDock and AutoGrid programs. Depending on the shell you are using, enter one of the commands below. You can see your shell type with the command echo $SHELL For bash users Type 'ulimit -s unlimited' and press <Enter>. For csh / kcsh users Type 'limit stacksize unlimited' and press <Enter>. 2. Fetch the protein-ligand completed 3PTB.pdb from Protein Data Bank (www.pdb.org), and save in the directory 'docking'. Now you need to extract the protein and ligand coordinates into separate files. Extract Protein and Ligand from the 3PTB.pdb 2.1.Using grep [user@host1 docking]$ grep ATOM 3PTB.pdb > protein.pdb [user@host1 docking]$ grep HETATM 3PTB.pdb | grep HOH -v | grep CA -v > ligand.pdb 3. Start the ADT 3.1.Type 'adt&' in the terminal and press <Enter>. After ADT has loaded uncheck the button 'float Camera' to join the viewer and the menu of ADT ADT is a viewer used to run docking jobs for AutoDock. Every structure in the ADT can be rotated using the middle mouse button, translated by pressing right mouse button and it can be zoomed by pressing 'SHIFT' button on keyboard and middle mouse button.

After ADT is loaded, begin the docking task The Docking Task 4. Preparing the Protein file protein.pdbqs 4.1.Open the protein.pdb file File > Read Molecule 4.2.Color the protein Color > By Atom Type A dialog box appears, check 'lines' and press 'OK' 4.3.Adding polar hydrogens to the protein Edit > Hydrogens > Add Choose 'Only Polar' from the dialog box and press 'OK' 4.4.Adding charges Edit > Charges > Add Kollman Charges Total charge added is desplayed in a dialog box. Press 'OK' 4.5.Check total charges on the protein Edit > Charges > Check Totals on Residues A dialog box appears displaying no residues with integral chrarge found. Press 'OK' 4.6.Adding solvent parameters and save as .pdbqs files Grid > Macromolecule > Add Solvent Parameters. A dialog box Is Molecule already in viewer? appears. Press 'Yes' and select the protein from the list. Press 'Select Molecule'. It will now prompt to save the file. Save it as protein.pdbqs in the directory 'docking'. 4.7.Remove the protein file from the viewer Edit > Delete > Delete AtomSet 5. Preparing Ligand file - ligand.pdbq 5.1.Opent the ligand.pdb files Ligand > Input Molecule > ReadMolecule Choos filetype as '.pdb' and read the ligand.pdb file. A dialog box displaying the information about ligand appears, press OK 5.2.Adding charges Edit > Charges > Compute Gasteiger Total charge added is displayed in a dialog. Press OK 5.3.Check total charges on the ligand Edit > Charges > Check Totals on residues A dialog box appears displaying no residues with integral charge found. Press 'OK' 5.4.Define Rigid root for ligand Ligand > Define rigid root automatically You can see a green sphere in the molecule which is defined as root. Root is the center about which the ligand is rotated during the docking procedure.

5.5.Define rotatable bonds in the ligand Ligand > Rotatable Bonds > Define Rotatable Bonds You can see the rigid bonds colored red and flexible or rotatable bonds colored green. Keep the default options and press 'Done'. 5.6.Setting the active torsions Ligand > Rotatable Bonds > Set number of active Torsions Keep the default values in the dialog box. Click on the text box and press <Enter>. Then press 'Dismiss' 5.7.Save the ligand.pdbq file in directory 'docking' Ligand > Write PDBQ Save this file as ligand.pdbq in directory 'docking' 5.8.Remove the ligand from the viewer. Edit > Delete > Delete AtomSet 6. AutoGrid 6.1.Select the protein files Grid > Macromolecule > Read Macromolecule Select the protein.pdbqs file from the directory 'docking'. A dialog box displaying the information about protein appears. Press 'OK' 6.2.Select the ligand file Grid >Set Map Types > By Reading Formatted Files Select the ligand.pdbq file from the directory 'docking' A dialog box displaying ligand atom types appears, keep the default values and press 'Accept' Now you can see the ligand in the active site of the protein. 6.3.Setting the grid box on the protein Grid > Set Grid You will see a dialog box showing the default dimensions of the grid box. For this docking you will set the grid box centered on the ligand in the complex. In the grid dialog box, Set X Y Z dimensions to 60 each Center > Center on ligand Visualize the modified grid box View > Show Box Toggle the show box button Save the grid settings File > Close saving current 6.4.Write the Grid Parameter file. Grid > Write GPF Save the file as protein.gpf in the direcotry docking 6.5.Start AutoGrid Run > Start AutoGrid In the dialog box press 'Launch' Autogrid will run in background and when it completes a message autogrid3:Successful completion will appear in the terminal from where ADT was launched.

Wait till Autogrid completes. 7. AutoDock 7.1.Select the protein Docking > Set Macromolecule > Choose Macromolecule Select the protein file from list and press 'Select Molecule' 7.2.Select the ligand Docking > Set Ligand Parameters > Choose Ligand Select the ligand file from list and press 'Select Molecule'. A dialog box displaying 'AutoDpf Ligand' parameters appears, keep the default values and press 'Accept'. 7.3.Set search parameters for docking' Docking > Set search parameters > Genetic Algorithm Parameters A dialog displaying the 'GA' parameters appears. Keep the default values and press 'Accept' 7.4.Set run parameters fro docking Docking > Set Docking Run Parameters A dialog displaying these parameters appears. Keep the default values and press 'Accept' 7.5.Write the docking parameter files Docking > Write DPF > GALS.dpf Save the file as ligand.protein.dpf 7.6.Start AutoDock Run > Start AutoDock In the dialog box press 'Launch' AutoDock will run in background and when it completes a message autodock3: Successful completion at localhost will appear in the terminal from where ADT was launched. Wait till AutoDock completes. 7.7.Delete the ligand file from the viewer Edit > Delete > Delete Molecule Select the ligand file from the list and press Delete Molecule. 8. Analyzing the docking results After Autodock compltes the docking results are saved in a file named ligand.protein.dlg in the directory 'docking' 8.1.Load the ligand.protein.dlg into the viewer Analyze > Docking Logs > Read Docking Log Select the ligand.protein.dlg file from the dorectory 'docking'. A dialog box displaying the total docked conformationa appears, press 'OK'. 8.2.Color the protein and ligand appropriated for clear visualization. Color protein in CPK and zoom the active site.

Select > Select From String In the dialog box, select 'protein' from the 'Molecule List' drop down and press 'Select'. You can see the protein selected in the viewer. Press 'Dismiss' to remove the

'Select From String' dialog. Un/Display > CPK In the dialog box decrease the 'Scale Factor' to 0.6, press 'OK' Color > By Residue Type > RasmolAmino In the dialog select CPK and press 'OK' Zoom the active site on the protein.

Display the ligand in Ball Stick Select > Select From String In the dialog box, deselect 'protein' from the 'molecule list' dropdown and select 'ligand'. Press 'Select'. You can see the ligand selected in the viewer. Press 'Dismiss' to remove the 'Select From String' dialog. Un/Display > Sticks and Balls In the dialog box leave the default values, press 'OK' Color > By Atom Type In the dialog select 'Balls and Sticks' and press 'OK' Press 'Clear Selection' in the main menu.

8.3.View the docked conformations Analyze > Conformations > Show Conformations You will see a dialog box with information about each docked conformation. Press the front arrow to scroll the conformations. You can simultaneously see the conformations in the viewer. The conformation with the lowest docking energy is ranked best by AutoDock. Make a note of the binding energy. 8.4.View Clustering Analyze > Clusterings > Show Clusterings You can see the clusters of docked conformations based on Binding energies. 8.5.Observing the docking energies of the ligand in .dlg file You can see the energies of the docked conformations in the .dlg file. Open and scroll down the protein.ligand.dlg file until you find 'CLUSTERING HISTOGRAM' Make a note of the cluster rank, lowest docked energy, number of conf. in the cluster.

Molecular databases for Drug design

In this exercise you will learn about the molecular databases used for drug design. Here is a brief introduction of two freely available databases, The ZINC database and the NCI database. 1. The ZINC Database ( http://zinc.docking.org ) The ZINC database is a collection of over 3.3 million compounds from various commercial and academic resources. ZINC is provided by the Shoichet Laboratory in the Department of Pharmaceutical Chemistry at the University of California, San Francisco (UCSF). At the ZINC database you can Browse / download subsets by vendor (provider), Browse / download pre-classified subsets specified by molecular properties, Search the database based on molecular constitution & properties and Upload your own molecules and define them as a set.

Right now molecules from 22 vendors can be downloaded from zinc. Each molecule in zinc has representation of its multiple protonation states and multiple tautomeric forms for each substance. 2. The NCI database The NCI database is a collection of 250,251 molecules publicly and freely available from NCI's Developmental Therapeutics Program (DTP). The entire collection with various biological activity information and calculated properties can be freely downloaded at http://cactus.nci.nih.gov/ncidb2/download.html The NCI database has an advanced browser to search the molecules. The advanced browser can be found at http://129.43.27.140/ncidb2/ .Using the advanced NCI browser you can search the database using various criteria like Substructure similarity, Exact similarity, Formula, Molecular weight etc., More documentation on how to perform queries and can be found at http://129.43.27.140/ncidb2/help.html

Advanced part Molecular Databases & In-Silico Virtual Screening

The aim of this part of exercise is to search for ligands with similar molecular constitution as the natural ligand with specified molecular properties as constraints using the interface at zinc database. Then you need to randomly select few hits (at least 5), dock them to Trypsin individually through the learnt procedure in the previous session (basic part) and rank the molecules according to the docking energies. This procedure if done at large scale in an automated fashion is called In-silico virtual screening. You need to use the macromolecule file protein.pdbqs from the previous practical session. 1. Performing search and Screening the hits Create a directory 'screening' by typing 'mkdir screening' in the home directory. NOTE: If you have problems in this section, you can copy the ligands.zip file at the course homepage and skip until 1.11 1.1.Open the web browser and visit the Zinc Database at http://zinc.docking.org Zinc is a free database of over 3.3 million commercially-available compounds for virtual screening. 1.2.Click on Search and Browse link to open the interface to search the database. This will open the search interface for the zinc database on right you can set the property constraints for the search and on right you can see a Java molecular editor in which you can draw the molecular constitution that you want to search. 1.3.Draw the natural ligand in the molecular editor. While drawing, observe that hydrogens are added automatically to the nitrogens. You can see the 2d structure of the natural ligand in the figure below. Now click on 'save SMILES' button just below the editor. NH2 will appear in the zinc molecular area on the nitrogen with the single bond in the figure below.

1.4.Specify the property constraints. Here we specify the constraints from Lipinsky's rule of five which is derived empirically from the analysis of the World Drug Index on properties that maximizes an oral drug candidate's probability of surviving clinical development. For this exercise we skip the partition coefficient constraint logp whose value should be less than 5. 1.5. Specify the following constraints Molecular Weight <= 500 # Hydrogen bond acceptors <= 10 # Hydrogen bond donors <= 5 # Rotatable bonds <= 10 1.6.Dont change any other values and click on 'QUERY DATABASE' button at the bottom of the page.

PS : DO NOT check the 'No time limit' dialog box If the server is not so busy then it should return the hits in few minutes.

1.7.On the results page you can see the ZINC ID, 2D structure of the hit, supplier, catalog number, and the molecular properties. Note: The format of the results might vary on the version of the database. It is updated often and so it might differ from what was explained above. 1.8.For every hit just below the supplier-catalog number, you can see various file formats in which the molecule can be saved. You can choose any of the formats but for now we choose SDF format. Choose at least 5 molecules from the hits and for each molecule click on SDF and save with zinc_id as the filename and extension .sdf (eg., zinc_305.sdf) in the directory 'screening'. 1.9.In the directory 'screening', make directories with zinc_id names for all the ligands downloaded from the zinc database. ( eg., 'mkdir zinc_305' ) 1.10. In order to perform the dockings for all these molecules we need the ligand in .pdbq format. For this we use the PRODRG server. The PRODRG server is used to generate the 3D coordinates and convert the molecules into various file formats. 1.11.

Open the PRODRG site at http://davapc1.bioch.dundee.ac.uk/programs/prodrg/ Here you can see a text area where you can paste the input file PDB, MDL MOL/SDF file formats. Open each of the downloaded ligands in a text editor (e.g., 'nedit zinc_305.sdf'), copy and paste in the text box. Select Chirality: YES, Full charges: YES and Energy minimization: YES and click on 'Run PRODRG' button. On the result page you can see various file formats that the molecule can be saved. Look for 'AUTODOCK PDBQ file (version 2.4, version 3.0)'. Click on version 3.0 Select all the text in the text box below The AUTODOCK 3.0 PDBQ file, copy, paste in text editor and save as a new file with name as zinc_id and extension .pdbq in the respective directories. (eg zinc_305.pdbq)

NOTE: In case the PRODRG server doesn't respond you can copy the ligands.zip file at the course homepage.

1.12. We have protein.pdbqs from the previous practical session (In-Silico Docking) in the docking directory. Copy the protein.pdbqs into all the directories in the screening folder. 1.13. For all the ligands perform the dockings as done in the previous practical session (In-Silico Docking) in the respective directories. a) Alterations during dockings

Skip 4. Preparing the protein file protein.pdbqs Skip 5. Preparing the Ligand file-ligand.pdbq since we already have the zinc_id.pdbq files from the PRODRG server. In 6.Autogrid at 6.3 setting the grid box on the protein, Set X Y Z dimensions to 60 each. DO NOT Center on ligand. Instead, specify the x center: -1.856, y center: 14.366 and z center: 16.748. This is because the ligand might be away from the protein initially and centering the grid on the ligand will exclude the protein from the docking.

1.14. zinc id

Note down the binding energies for each ligand and tabulate the results Molecular wt. Docking Energy # of conf, in top cluster

Make a note of the ligand with lowest binding energy.

-- Chaitanya, Francesco & P G Nyholm, Biognos AB