I. .Basic Operations of SYBYL

II. Basic Docking

III. Building an Inhibitor

IV. Advanced Docking

The SYBYL package is optimized for operations with small molecules and their interactions with biopolymers. The program operation is similar to the InsightII and is menu-driven using an intuitive command structure. We will first practice some basic operations that allow you to manipulate biopolymers and their complexes.

Read

Select 1AQ7.pdb (This file contains the structure of an inhibitor bound to trypsin). (Read about this structure here)

Click OK

A dialog box appears asking "Center the molecule"

Click OK

The molecule is now displayed on the screen. You can control the size and orientation with the mouse buttons identically to how you do it in InsightII.

Use the "center-right" mouse buttons to fit the entire molecule to the screen size

We will now center the rotation axis around the inhibitor.

Center

Click on the "Sets" button

In the new window, Click on the UNK_ATOMS

Then click OK in the two windows.

We will now 'undisplay' the water molecules.

Undisplay Atoms

Click on the "Sets" button

In the new window, Click on the Water

Then click OK in the two windows.

Now we will differentially color the enzyme and the inhibitor

Color

Atoms

Click on the "Sets" button

In the new window, Click on the UNK_ATOMS

Then click OK in the two windows.

A new window appears. Select a color (Red is good)

Color

Atoms

Click on the "Sets" button

In the new window, Click on the UNK_ATOMS

Click OK in this window.

In the selection window, click on the 'INVERT' Button

Click OK in this window.

A new window appears. Select a color (Green is good)

Now we will display the different parts of the enzyme active site: the residues responsible for substrate binding specificity and catalytic activity

Color

Atoms

Click on the "Substructures" button

Click OK in this window.

Then click OK in the two windows.

A new window appears. Select a color (Yellow is good)

We have just highlighted the residue that recognizes the positively charged side chains in trypsin substrates and inhibitors. Now we will examine the "catalytic triad"-i.e., the residues that perform the actual bond cleavage in all serine protease.

Color

Atoms

Click on the "Substructures" button

Click OK in this window.

Then click OK in the two windows.

A new window appears. Select a color (White is good)

You can now use the mouse buttons to move the molecule and examine the interaction of the inhibitor with the enzyme-its binding site and why it functions as an inhibitor.

Now we will learn basic docking techniques. Before doing that we will have to clear the screen.

Zap/Delete Molecule

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Read

Select 1AQ7.pdb (This file contains the structure of an inhibitor bound to trypsin)

Click OK

A dialog box appears asking "Center the molecule"

Click OK

Center the rotation axis around the inhibitor.

Center

Click on the "Sets" button

In the new window, Click on the UNK_ATOMS

Then click OK in the two windows.

In order to work with one molecule in the absence of another, the molecules must be in different work areas. The trypsin molecules and the inhibitor are stored in the same file in the database, so before we can dock the inhibitor, we have to extract it.

In order to extract a molecule, we have to have somewhere to put it. SYBYL has an option that allows you to divide the screen into several work areas, each of which can hold different molecules. We will start by dividing the screen into halves

On the left side of the SYBYL main window there is a series of buttons. The third one down looks like this: .

Click the button and then click the "Half" button, followed by the Q (quit) button.

Copy the cofactor to another molecule area

Extract

Click on the "Sets" button

In the new window, Click on the UNK_ATOMS

Then click OK in the two windows.

A new window appears.

Select M2:<empty> as the molecule area

Click OK

Now we will delete the inhibitor from the original complex.

Delete

Click on the "Sets" button

In the new window, Click on UNK_ATOMS.

Then click OK in the two windows.

Now we will name our inhibitor so that it will be easier to keep track of.

Name molecule

Select M2

Click OK

Type inhibitor in the name block

Click OK

The docking process evaluates the suitability of an individual position/orientation by evaluating the interaction energies. As you already know, hydrogen bonds and charge interactions are important in molecular interactions. Thus, we must prepare the molecules to have the appropriate charges and numbers of hydrogens.

The first step in adding hydrogens is to ensure that SYBYL understands what kind of bonds are connecting the atoms in the molecule of interest.

Modify

Bond

Select 'Type' as the element to modify.

Click OK

Select M2:inhibitor as the molecule

Then press the Bond Types button

In the next window, select uk (unknown

Click OK

Click OK in the Bond Expression window

The bond between the oxygen and the benzene moiety is highlighted in green, and a new window appears with a series of bond types 1 (=single), 2 (=double), etc.

Select 1 and click OK

The window disappears indicating that SYBYL understands all the other bond types in the molecule.

Now we will actually add the hydrogens.

Add

Hydrogens

Select M2:inhibitor in the molecule area window

Click OK

Hydrogens are now added to the inhibitor.

Now we must assign the partial charges to the inhibitor atoms.

Charges

Gasteiger-Hò ckel

Select M2:inhibitor in the molecule area window

Click OK

A dialog box appears asking whether you want to change formal charges before computing charges

Select NO

Before beginning the docking process, color the molecule as we had done before.

Color

Atoms

Click on the "Sets" button

In the new window, Click on the UNK_ATOMS

Click OK in this window.

In the selection window, click on the 'INVERT' Button

Click OK in this window.

A new window appears. Select a color (Green is good)

Color

Atoms

Click on the "Substructures" button

Click OK in this window.

Then click OK in the two windows.

A new window appears. Select a color (Yellow is good)

Color

Atoms

Click on the "Substructures" button

Click OK in this window.

Then click OK in the two windows.

A new window appears. Select a color (White is good)

There is a problem with the default settings of SYBYL. Before we can start the Docking procedure, we have to fix it.

Tailor

Set

Select Dock in the Options window-Click OK

Select MAX_LATT_POINTS in the Tailor Option window-Click OK

A new window appears:

Change the value from 200000 to 400000-Click OK

Click END in the two Option windows

Now we are finally ready to begin docking!

To facilitate the docking, change the screen back to a single window:

Click the button and then click the "Full" button, followed by the Q (quit) button.

Dock

Select M2:inhibitor as the Ligand Molecule

Click OK

Select M1: as the Site Molecule

Click OK

Three new items appear on the screen.

• An energy display box, showing the total energy of interaction, plus steric and electrostatic components.
• A box surrounding the ligand interaction site.
• A terminal window at the bottom of the screen.

Ordinarily, mouse movements move both the ligand and the target together. To have the mouse control only the ligand, press the F10 key on the keyboard

Now the translations and rotations only affect the ligand's movements.

Slowly move the ligand around and attempt to bring it into an energy minimum.

When you are done, exit back to the main menus by placing the cursor in the terminal window and pressing the ENTER key.

To return the appropriate mouse controls, click the button on the left row of buttons and select Global followed by the Q (quit) button.

To be sure the program behaves properly, we must 'reset' it. Do this by clicking the 'Reset' button on the left row of buttons and then click the 'Everything' button followed by the Q (quit) button.

Now we will learn how to build our own inhibitor and dock it into trypsin. Before doing that we will have to clear the screen.

Zap/Delete Molecule

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We will be building a benzene-based inhibitor of trypsin from benzene and arginine. (Read about the properties of this inhibitor here)

Get Fragment

Scroll down and select Benzene from the option list

Click OK

Get Fragment

Scroll down and select Arginine from the option list

Click OK

Select M2 as the molecule area

Click OK

We will now edit the arginine molecule to obtain the guanidine groups.

To move the arginine out where it can be operated on, click F10, and use the mouse to move the group out of the way.

To make life easier in terms of knowing which atoms to delete, we will label them.

Label

Atom name

Click on M2 in the Atom Expression window

Click the Atom Types button

In the next window, select C,O,N

Click OK

Click OK

Now we will create the guanidium group

Delete

Bond

Click on the N2 nitrogen and the C5 carbon in the molecule display window.

Click OK

Now we will delete the "remainder" of the arginine residue.

Delete

Substructure

Click on the Arg side chain in the molecule display window

Click OK

Now we will prepare for the 'chemical' joining of the benzene and the guanidium group.

Delete

Atom

Click the proton on the N2 nitrogen in the molecule display window.

Click OK

Now join the benzene and guanidinium moieties.

Other Tools

Join molecules

When the select Target window appears, click on one of the benzene protons

Repeat this procedure placing the guanidinium group on the benzene at a para position with respect to the first guanidinium group.

The final molecule will look like this:

Now we will save this inhibitor

Save As

Select Benzene

Call the file inhibitor1

Click OK

Zap/Delete Molecule

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Now we will dock our inhibitor into the trypsin molecule.

Read

Select 1AQ7.pdb (This file contains the structure of an inhibitor bound to trypsin)

Click OK

A dialog box appears asking "Center the molecule"

Click OK

Delete

Atom

Click on the "Sets" button

In the new window, Click on the Water

Then click OK in the two windows.

Now we will add H to the protein

Add Hydrogens

Click on the All button

Click OK

In Option window (which hydrogens to add), select All

Click OK

Now we will load the protein's partial charges

Load Charges

Click on the All button

Click OK

In Option window accept the Koll_Uni default

Click OK

To facilitate building of the new inhibitor complex, we will superimpose the new inhibitor on the old one and then delete the old one and begin the docking procedure.

Undisplay Atoms

Click the Sets button

Click OK

Click the Invert button

Click OK

Now read in the new inhibitor

Read

Select inhibitor1

Click OK

To help distinguish one from the other color the new inhibitor cyan

Color

Atoms

Click on M2

Click OK

Choose cyan and click OK

To help you in aligning the correct atoms, label the molecules

Label

Atom name

Click on M1 in the Atom Expression window

Click the Atom Types button

In the next window, select C,O,N

Click OK

Click OK

Repeat this procedure using M2 as the atom option.

Try using to align the one of guanidinium groups of the new inhibitor the guanidinium group o the old inhibitor. Use the F10 key to toggle mouse control between the new inhibitor and both molecules to aid this process.

Now we will re-display the protein.

Display Atoms

Click on M1

Click on the All button

Click OK

Now delete the old inhibitor

Delete

Atom

Be sure M1 is selected

Click on Set

Select UNK_ATOMS

Click on OK in this and previous window.

Now Dock your inhibitor using the Docking procedure above.

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