Ligand-Protein Docking

Computer Aided Drug Design Practical for

3rd Year Pharmacy Students

SYBYL 7.0 Basics

• Starting Sybyl
  • sybyl

   

• Menubar
  • SYBYL's menubar is similar to that of a PC application's menubar. The presence of an active (black font) SYBYL menubar indicates that the program is waiting for your menu selection.
  • A menu item can be a simple command or a check box; it can also lead to a submenu or a dialog. Additional categories may be available if you have licensed special modules.
  • By default, all options are shown in the menus. Any options for which you do not have a license are greyed out (these most likely will occur in the Tools menu).

   

• Toolbox Icons
  • The SYBYL tools used to interact with the graphics are represented by icons along the left edge of the SYBYL screen.
  • To activate an icon, place the pointer on it and press the left mouse button. In cases where a dialog is displayed, you can close the dialog by pressing the Q button.
  • From the top to bottom, the graphics icons are:
    Support Information, Mouse Focus, Display Options, Display Area Options, General Structure Display, Graphic Attributes, Reset, Rotatable Bonds, Z-Clipping, Depth Cue, Restack, Stereo Adjustment, Physical Dial Box, Virtual Dial Box, Extents and Rate Mode.
     
• Textport Window
  • The textport window enables you to enter any command, including HELP. Review the Tripos Bookshelf for a complete listing of all SYBYL commands available. After entering a command, the prompt is again printed on the terminal.
  • Note: When running SYBYL, both menu selections and command line entry are available simultaneously.

   

• Help
  • The Tripos Bookshelf is SYBYL's online help tool. This interactive documentation enables you to view Tripos documentation online. It is context sensitive and is a valuable tool to help you learn more about SYBYL.
  • The Tripos Bookshelf contains:
    Index, Connection to Tripos web site, Full color pictures, FAQs, Search functionality, Citations, Articles, Work flow charts, PDF library.
     
• Mouse Buttons
  • The left button is used to select SYBYL menu items/objects (by picking atoms), to open/close graphics icons, and to select items in the dialogs.
  • Pressing mouse button combinations and moving the mouse cause various motions of objects in the "active" display area, below is a summary table:

Mouse button	Function
left	selection
mid + right	scale
right	X-Y rotation
left + right	Z rotation
mid	X-Y translation

Working with Molecules

• Load Molecules from the Fragment Database
  • Build/Edit >>> Get Fragment
  • Example:
    • Load 1,3-DIOXANE into SYBYL.
      • Build/Edit >>> Get Fragment.
      • Select 1,3-DIOXANE.
      • Press OK.
      • The molecule loads into SYBYL's display area 1, also known as D1. Its default location is in molecule area 1, also known as M1.
        • A molecule area is a region of memory that holds a particular molecule. Molecule areas do not have a defined number (the number depends on the computer's memory). You can place hundreds (if not thousands) of molecule areas in a display area on a standard workstation.
        • A display area is where your molecule is going to be displayed on the screen. SYBYL has four unique display areas: D1, D2, D3, and D4.
        • Note that molecule areas have rules:
          
          M1 is always in display area D1.
          M2 is always in display area D2.
          M3 is always in display area D3.
          M4 is always in display area D4.
          
          If additional molecule areas are used, they simply recycle through the display areas:
          
          M5 is always in display area D1.
          M6 is always in display area D2.
          M7 is always in display area D3.
          M8 is always in display area D4.
    • Change the characteristics of your loaded molecule.
      • Press .
        The Display Options dialog is displayed. This dialog enables you to modify the various look of your display area and molecules.
      • In the Display Options dialog, select Sticks.
        The lines making up your molecule become thicker and the color codes are more defined. From now on, if you load any molecule in SYBYL, it will load in Sticks mode.
      • Press Q to close the Display Options dialog.
    • Load another molecule into SYBYL.
      • Build/Edit >>> Get Fragment.
      • Select 1,2,4-Trioxolane.
      • Press OK.
      • M1 is already being used by 1,3-dioxane. However, the other molecule areas (M2 - M10) are currently empty. You must choose which molecule area should house the newly loaded molecule.
      • In the Molecule Area dialog, select M2:<empty>.
      • Press OK.
      • You have now placed two molecules within SYBYL (1,3-Dioxane and 1,2,4-Trioxolane). The two molecules reside in two unique molecule areas (M1 and M2).
• Read/Write Files of Molecules
  • Read in Structure from File via the Menubar
    • To open a file for reading, select File >>> Read.
    • The Read File dialog uses the filename as the default for the molecule name when reading a single file. If any form of molecule name is present in the input file, that name is used instead.
  • Write out Structure from File via the Menubar
    • Select File >>> Save As.
    • Specify Format.
    • Multiple structures can be selected from the list and saved to a MOL file, MOL2 file, SD File, or SLN file. Use the Select All, Invert, and Clear buttons to manipulate the selections in the molecule list.
      • MOL2 files are ASCII files containing all information necessary to reconstruct the molecule. The format is based upon the convention of a keyword for each type of data needed to reconstruct the molecule, followed by a group of records.
      • A MOL file does not preserve the complete information available with molecules in SYBYL 6.x and above. It is provided for compatibility with existing user programs. Data written to a MOL file include atoms and bond information, rotatable bond definitions and plane equations. To preserve the complete molecular description, use the MOL2 file format.
• Rotate the Molecules
  • Use the Mouse Focus Option dialog to differentiate between the two molecules that were previously displayed:
    • The display area is set to Global by default when you start a SYBYL session. All images on the screen -- molecules and backgrounds -- are affected simultaneously by rotations and translations.
    • Rotate the molecules by pressing the right mouse button and moving your mouse in any direction.
    • Notice that both molecules move. This is because your current molecule setting is on Global (global).
  • Move 1,3-DIOXANE to the upper right corner:
    • Press the button.
    • Select D1 in the Mouse Focus Options dialog.
      By selecting D1, you are telling SYBYL to focus in on 1,3-DIOXANE.
    • Press the middle mouse button and move 1,3-DIOXANE to the upper right corner of SYBYL.
  • Move 1,2,4-TRIOXOLANE to the upper left corner:
    • Unselect D1 and select D2 in the Mouse Focus Options dialog.
      By deselecting D1 and selecting D2, you are telling SYBYL to focus in on 1,2,4-TRIOXOLANE.
    • Press the middle mouse button and move 1,2,4-TRIOXOLANE to the upper left corner of SYBYL.
• Clear the Screen
  • Build/Edit >>> Zap (Delete) Molecule
  • ZAP deletes molecules and their associated data structures from program memory. It clears the molecule area. All associated display structures (e.g., dots, ribbons, ...) are removed from the graphics screen as well.

Protein

1. Load a protein (HIV-1 protease).
   • File >>> Read.
   • Select protein.pdb and press OK.
   • Select YES in the Center the molecule dialog and press OK.
2. Name the protein.
   • Build/Edit >>> Name Molecule.
   • Type HIV_protease for the new molecule name (press OK).
3. Add hydrogens to the protein.
   • Biopolymer >>> Prepare Structure >>> Add Hydrogens.
   • Press OK.
4. Compute charges for this protein.
   • Biopolymer >>> Prepare Structure >>> Load Charges.
   • Press OK.
   • Press Yes.
5. Save the protein
   • File >>> Save As.
   • Type hiv_protease for the new molecule name (press Save).
6. Zoom out to view the entire protein.
7. Find secondary structures in protein.
   • Biopolymer >>> Conformation >>> Find Secondary Structure.
   • Use the Kabsch-Sander method.
   • Select Render Conformations and press Find.
   • Press Close.
8. Construct a three-dimensional shaded ribbon for the protein.
   • View >>> Biopolymer Display >>> Shaded Ribbon.
   • Press All and press OK.
   • Select WHITE as the color and press OK
9. Highlight catalytic triad ASP25, THR26 and GLY27 by spacefill representation.
   • View >>> Mixed Rendering.
   • Press Substructures and select ASP25, THR26 and GLY27 from both chain A and B and press OK.
   • Press OK.
   • Select SpaceFill for Representation and press OK.
10. Question 1: Where is the active site of the protein?
11. Delete the protein.
    • Build/Edit >>> Zap (Delete) Molecule.

Ligand

1. Load a drug (A77800).
   • File >>> Read.
   • Select ligand.pdb and press OK.
   • Select YES in the Center the molecule dialog and press OK.
2. Name the ligand.
   • Build/Edit >>> Name Molecule.
   • Type A77800 for the new molecule name (press OK).
3. Add hydrogens to this drug.
   • Build/Edit>>> Add >>> Hydrogens.
4. Compute charges for this drug.
   • Compute >>> Charges >>> Gasteiger-Huckel.
   • Press No when asked if you want to change formal charges before computing charges.
5. Conduct molecular mechanics computation to find lowest potential energy conformation for this drug.
   • Specify the force field and type of charges to use.
     • Compute >>> Minimize.
     • In the Minimize dialog, press Modify.
     • In the Energy dialog, select Tripos from the Force Field option menu.
     • Select Gasteiger-Huckel from the Charges option menu.
     • Press OK.
   • Set the minimization parameters.
     • In the Minimize dialog, press Minimize Details.
     • In the Minimize Details dialog, increase the maximum number of iterations (Max.Iterations) to 10000.
     • Decrease the Gradient to 0.005.
       The Gradient value is a termination criterion. If the gradient difference between two consecutive iterations goes below this value, the calculation stops.
     • Set the color option to Force.
       This color codes atoms according to the total force on each atom, as the minimization proceeds.
     • Press OK.
   • Submit the job to run interactively.
     • In the Minimize dialog, type A77800 as the job name.
     • Press OK.
       As the minimization proceeds, changes to the structure are interactively displayed. Information about each iteration is also printed to the textport.
     • Question 2: What is the total energy of the minimized structure?
6. Save the drug
   • File >>> Save As.
   • Type A77800 for the new molecule name (press Save).
7. Create molecular surface of this drug.
   • Create a molecular surface.
     • View >>> MOLCAD Surfaces >>> Molecular Surfaces
     • Press Create in the MOLCAD Surfaces dialog.
   • Select type of molecular surface.
     • In the Create MOLCAD Surface dialog, select Connolly from the Type menu.
     • Press OK.
     • Press Done.
8. Delete the drug.
   • Build/Edit >>> Zap (Delete) Molecule.

DOCK

SYBYL's docking functionality provides a real time approximation of the intermolecular energy of interaction between a pair of molecules (in kcals/mol), a useful tool for interactively identifying possible binding conformations. One molecule (stationary, by convention) is called the site, and the other is the ligand. Interactive output includes the total energy, the magnitude and direction of the overall force (for ligand atoms strongly interacting with the site), and the one site atom which is interacting most strongly.

1. Load protein (hiv_protease.mol2) and drug (A77800.mol2). It is recommended that protein be loaded first.
2. Position the ligand into the active site of the protein (you may wish to turn on shaded ribbon for the protein and spacefill for ligand).
3. Prepare DOCK parameters.
   • Options >>> Tailor.
   • In the Tailor dialog, select DOCK from the Subject menu.
   • Increase the maximum number of lattice points (MAX_LATT_PTS) to 800000.
   • Press Apply and then press Close.
4. Begin docking.
   • Compute >>> Dock.
   • Select M2:A77800 as the ligand molecule area (press OK).
   • Select M1:HIV_protease as the site molecule area (press OK).
     Three new items appear on the screen:
     1. An Energy Gadget dialog, showing the total energy of interaction, plus its steric and electrostatic components, for the currently displayed configuration using the Tripos force field.
     2. A box surrounding the ligand molecule. As you move the ligand, the energy of interaction is computed only for atoms within that box.
     3. A prompt at the command line for the next action SYBYL is to take.
        • press <return> to exit the DOCK command;
        • press ? to show other actions that can be taken from within the docking process.
5. Move the ligand.
   • Slowly move the ligand a few Å in any direction.
     As an atom of the ligand gets too close to a site atom, the energy of interaction suddenly increases, and new yellow and red lines are displayed.
     • Yellow lines connect ligand atoms to the nearest bumping site atom,
     • Red lines show the direction that a ligand atom should be moved to reduce the repulsive energy.
6. Minimize docking energy
   • With the textport active, type MINIMIZE_DOCK (press the return key).
   • Select SITE as the molecule to have fixed geometries (press OK).
   • Wait for minimization to finish running.
   • Question 3: What is your final docking energy?
7. End the docking operation.
   • When finished docking, exit back to the main menu by pressing <return> in the textport.
8. Warning: If you exit docking mode, then re-enter it, you may get an error message regarding a lack of memory. This is due to a memory allocation problem that cannot be avoided. The only work-around is to exit SYBYL and restart. Freeze the current view and save it to a database if you want to be able to restart exactly where you left off.

FlexiDock

Genetic algorithm-based Flexible Docking provides a means of docking ligands into protein active sites. FlexiDock works in torsional space, keeping bond lengths and angles constant. As large vdW interactions can only relax via bond rotation(s), optimization cannot alter chiral centers and bond stereochemistry. FlexiDock works on a protein/ligand pair. The protein backbone atoms are fixed in space, but the ligand is mobile (rotation/translation can be applied). Both the protein (sidechains only) and the ligand can contain a number of flexible bonds. However, to speed up calculations, FlexiDock considers only non-ring single and amide bonds as rotatable.

FlexiDock uses a genetic algorithm to determine the optimum ligand geometry. Genetic algorithms are relatively robust global optimizers, with performance requirements which scale well with increasing system size. The fitness function uses a subset of the Tripos force field: the van der Waals, electrostatic, torsional and constraint energy terms, and calculates the energy of the important atoms in the supermolecule.

1. Set Up FlexiDock Structures
   • With the ligand docked inside the protein's active site,
     • Compute >>> FlexiDock >>> Create an Input File.
     • In the Molecule Area dialog, select M1:HIV_protease, then press OK.
       The Set Up FlexiDock Structures and  Protein Display Options dialogs are then posted.
   • Protein Display Options dialog.
     • Define and visualize the protein binding pocket.
     • Press Define Pocket
     • In the Atom Expression dialog, replace M1(0) with M1(957) (press OK).
     • Increase the radius (Radius to Show Around Pocket) to 10.
   • Set Up FlexiDock Structures dialog.
     • The following seven items in this dialog correspond to the seven steps generally necessary to prepare a protein file from a structural database for submission to FlexiDock, and should be executed in the order suggested. To enforce this the check boxes on the left are organized as a cascade:
       • All are grayed out initially.
       • The first check box becomes accessible only after you have specified the location of the protein and ligand molecules, and both are in separate work areas.
       • Toggling on the first check box activates the next line, and so on.
       • Toggling off any check box turns all the lines below it off and disables them.
     • Five of the check boxes have associated action buttons to Remove the water molecules, Add the hydrogens, Add the atomic charges, Choose the rotatable bonds, and Choose H-bond sites. Each Choose button launches a subsidiary dialog for carrying out the corresponding operation.
       • To perform the desired operation, simply press the button. When the operation is completed, the check box is automatically toggled on and the next line becomes active.
       • If an operation is not necessary, simply toggle on the corresponding check box so you can proceed to the next operation.
     • Two of the check boxes have associated Hint buttons to help you perform the corresponding operations manually: checking the atom types and positioning the ligand in the cavity.
     • The two Hint buttons differ from the four action buttons. Pressing them does not toggle on the associated check box, nor does it activate the next lines.
     • For Water has been removed, press Remove to delete all the water molecules.
     • Check box for Atom types have been checked.
     • For Hydrogens have been added, press Add to fill all valences with hydrogens.
     • For Charges are in place, press Add to compute all atomic charges.
     • For Rotatable bonds are set, press Choose.
       • In the Pick Rotatable Bonds dialog, press All and press OK.
     • For H-bond sites are marked, press Choose.
       • In the Defining H-Bond Sites dialog, press Add, then All and OK for every item.
     • Check box for Ligand is pre-positioned in cavity.
     • Type hiv for file name and press Write Out FlexiDock Input File.
2. Run FlexiDock.
   • Press FlexiDock It.
     In three successive dialogs you will be prompted to specify:
     • The template parameter file to use. By default, this file is $TA_MOLTABLES/FlexiDock.par.
     • The random number seed to start from.
     • The maximum number for generations to allow. The default is 3000.
   • Accept all default values for the three dialogs.
   • Wait for FlexiDock to finish running.
   • Close Flexidock window by pressing Quit.
   • Delete both protein and drug.
3. View docking results.
   • Open spreadsheet.
     • File >>> Molecular Spreadsheet >>> New.
     • Select Database and press OK.
     • Select hiv.mdb and press Open.
   • Remove unnecessary information.
     • Select the first row.
     • MSS: Edit >>> Delete.
   • Import list of docking energies.
     • MSS: File >>> Import.
     • Select Custom in the Format option menu of the Import dialog.
     • Press the [...] button to its right to bring up the Custom Format dialog.
     • Select Space in the Delimiter option menu and toggle both Labels check boxes on. Enter * and NEW, respectively, in the row and column fields. This means that when the data file is imported its rows will match the existing rows and new columns will be created.
     • Press OK to exit the Custom Format dialog.
     • Enter hiv.mdb/hiv.txt in the File field.
     • Press Import in the Import dialog.
4. View docked structure.
   • File >>> Database >>> Open.
   • Select hiv.mdb and press Open.
   • Select READONLY as the access mode (press OK).
   • File >>> Database >>> Get Molecule.
   • Select HIV0001 (or select the molecule with the lowest energy).
   • Press OK.
   • Question 4: Is there any difference between the docked conformation and the initial conformation?
5. Delete all molecules.
   • Build/Edit >>> Zap (Delete) Molecule.
   • Press All and press OK.

INVDOCK

A small molecule is flexibly docked into a cavity by a procedure involving multiple-conformer shape-matching alignment of the molecule to the cavity followed by molecular-mechanics torsion optimization and energy minimization on both the ligand and the binding region of the receptor. A new scoring method is used that performs binding competitive analysis in addition to the evaluation of molecular mechanics ligand–protein interaction energy.

1. View docking results.
   • Open spreadsheet.
     • File >>> Molecular Spreadsheet >>> Open.
     • Select invdock.mdb as the Sub-Directories.
     • Select ranklist.tbl and press Open.
   • E is the total energy, Ev is the van der Waals energy, Eh is the hydrogen bonding energy, Ee is the electrostatic energy and Es is the steric energy.
2. View docked structure.
   • File >>> Database >>> Open.
   • Select invdock.mdb and press Open.
   • Select READONLY as the access mode (press OK).
   • File >>> Database >>> Get Molecule.
   • Select 1HIV0000101 (or select the molecule with the lowest energy).
   • Press OK.
   • Question 5: Is there any difference between this docked conformation and the docked conformation from FlexiDock?
   • Question 6: Is it valid to compare the energies of the docked conformations from DOCK, FlexiDock and INVDOCK? Why?