Cerius²·Ludi

6.2       Tutorial lesson 2: modifying a known inhibitor for HIV protease

Return to Tutorials contents.

Ludi can be used to suggest modifications for a known ligand. When run in link mode Ludi fits fragments in a way that simultaneously exploits available hydrogen-bonding and hydrophobic sites on the receptor and suggests how fragments might be bonded to the existing ligand. Ludi requires only the structure of the receptor, the structure of the inhibitor and a coordinate in the active site.

In this tutorial you will use Ludi to search for fragments that can be linked to a known HIV protease inhibitor to make a new inhibitor.

Tutorial

• Running Ludi in link mode.

• Loading Ludi hits.

• Reviewing hits.

• Linking a hit to the known ligand.

To start the tutorial select the File/Load Model... command, set File Format to MSI, go to the Cerius2- Resources/LUDI directory and double click first hvp.msi and then 1682.msi.

The models will load one-by-one in the Model window.

In the Visualizer, select hvp's diamond-shaped currency button and set l682 to visible by toggling its visibility button. Now select the Select BORDER Display button. The Select BORDER Display button is the last of the three display mode buttons on the upper right of the Visualizer.

2.   Viewing the ligand in the receptor site

The molecules displayed on the screen are HIV protease (lower left) and a modified version of the Merck Inhibitor L-682,679 (upper right).

The heavy atom coordinates for HIV protease come from entry 4hvp in the Brookhaven Protein Data Bank. The inhibitor MVT-101 and waters in 4hvp have been removed. The Merck inhibitor was built into the active site by analogy with MVT-101. Hydrogens have been added.

In this tutorial, Ludi will suggest molecules that can be bonded onto the Merck inhibitor to enhance its binding to the receptor.

Go to the Ludi module by clicking the List of Menu Decks in the upper-right quadrant of the Visualizer and selecting Ludi from the popup list.

The Ludi deck of cards will appear containing the ACTIVE SITE VIEWER, LUDI LIBRARY, ANALOGS BASED DESIGN and RECEPTOR BASED DESIGN cards.

3.   Specifying the Ludi library

Go to the LUDI LIBRARY card and click Library Spec.

The Ludi Library Specification panel will appear.

Each library comprises two files, a structure file, containing atomic coordinates, and a targets file, describing the interaction sites of the fragments.

Note that Ludi Library Specification panel is divided into two sub-panels, one for each of the two types of Ludi libraries: the De Novo Library which is used when Ludi is run in standard (de novo) mode and the Link Library which is used when Ludi is run in link mode.

In this tutorial you will use a link mode library designed specifically for the tutorials.

Set the Library Type under Link Library to User. Find ludi_tut_link_lib.str in the listbox under Cerius2-Resources/LUDI and click SELECT.

At the end of the tutorial, the File/New Session... command is run to reset the library to the default. If you quit the tutorial before its natural end, you should do this yourself before doing any other Ludi runs.

4.   Setting the Ludi preferences

Go to the RECEPTOR BASED DESIGN card and select Preferences.

The Ludi Runtime Parameters panel will appear.

For the tutorial the parameters are set to their default values: Interaction Site Parameters: Preselect 2.00 Density of Lipophilic Sites 25 Density of Polar Sites 25 Aliphatic_Aromatic off Fitting Weights: Link Weight 1.00 Lipo Weight 1.00 Polar Weight 1.00 Hit Criteria: Reject_Bifurcated off No Unpaired Polar on Electrostatic Check on Minimum Distance 2.5 Minimum Separation 3.00 Minimum Surface 0 Minimum Score 0 Maximum Unfilled Cavity 0 Maximum Hits 940

Please refer to the help text for descriptions of the parameters.

5. Running the Ludi Job

You are now ready to perform the Ludi run.

On the RECEPTOR BASED DESIGN card, click Find Hits.

The Ludi Receptor Based Design panel will appear.

Click the Defined Current Model as Receptor button to initialize hvp as the receptor. Select l682 by clicking just to the left of its ID number in the Visualizer Models panel. Check Link Sites and click Define Single Links from Selected Model to establish that each fitted fragment must be aligned with at least one linkage site on the ligand.

To fill the Search Sphere Center Coordinates , make l682 current and select a hydrogen atom on the nitrogen at the bottom of the screen. If you have selected the correct atom, the textport should read: Model(name=l682):Atom(id=40) Now click Define Center From Selected Atoms.

Set Search Sphere Radius to 5.00 Å and Maximum RMS to 0.5 Å.

To begin the Ludi calculation, click Find Ludi hits on the Ludi Receptor Based Design panel.

Open the Job Control panel by clicking the Job Control menu item on the RECEPTOR BASED DESIGN card. Click the Update button and then click Monitor Logfile.

This will open an xterm window displaying the log file. At the end of the run, the logfile will report that six hits were found. If you get an error message that says the logfile does not exist, wait a few moments and try the Monitor Logfile button again.

5.   Loading the results of the Ludi run

Proceed with this step only after the log file shows that the run is complete.

Click Load on the RECEPTOR BASED DESIGN card. When the Ludi Load panel appears, find the .run file for the run name noted on the Ludi Job Control control panel (you may have to hit the update button to see the most recent job) and click SELECT. Load the hits into the Ludi Score Table by clicking the Load button (make sure that the Hits parameter is toggled on). Note that you have the option (unchecked by default) of loading the results into a QSAR study table. This tutorial will assume that you are viewing the results in the default Ludi Score Table and not the study table.

The Ludi Score Table will appear listing the six hits.

If all the scoring information is available (This is true if the run was not killed and if you have not deleted files from the run directory.) the fragments are loaded by score from highest to lowest. If the scoring information is not available, the fragments are loaded in the order in which they were found in the Ludi library.

6.   Reviewing the results of the Ludi Run

The following commands will allow you to better view the active site and the hits.

Make hvp current and select all hvp atoms by placing your cursor next to it and dragging a box around it. Click Hide Hydrogen Atoms on the Atom Visibility control panel to hide all hydrogen atoms (the Atom Visibility control panel is accessible by selecting View/Atom Visibility... in the Visualizer).

Make l682 current and visible (i.e., make sure that its diamond-shaped currency button is selected in the Visualizer and turn off trypsin's visibility button).

Select all of the atoms in l682 by placing your cursor next to it and dragging a box around it. Now, turn l682 yellow by selecting View/Colors... in the Visualizer and changing Pen to YELLOW in the upper left of the Color Selected Objects control panel.

Now select the Select OVERLAY Display button. The Select OVERLAY Display button is the first of the three display mode buttons on the upper right of the Visualizer. Make sure both of the molecules' visibility buttons are selected.

It should be easier for you to see l682 in hvp. If not, try another color.

Make the first hit current by selecting its diamond-shaped currency button.

Now view each hit in turn by making it the current model.

It is worth noting that Ludi found fragments that align with two different bonds in the inhibitor. This happened since there was no restriction as to where a link could be made.

You may also specify the sites on the ligand at which link fragments are to be fit. To illustrate this the previous run will be repeated using a link site specification.

7.   Specifying the ligand link site

To prepare to specify the link site at which Ludi should fit fragments, select the ligand hydrogen atom as above.

On the Ludi Receptor Based Design control panel, check Link Sites and click Define Links from Selected Atoms to establish that each fitted fragment must be aligned with the specified linkage site.

To begin the Ludi calculation, click Find Ludi hits on the Ludi Receptor Based Design panel and monitor job completion from the logfile, as above.

8.   Loading the results of the second Ludi run

This time the logfile will report that five fragments have been found. Fewer fragments are expected in this case since Ludi is now only fitting fragments at one link site.

Load the hits found as above.

9.   Bonding a Ludi hit to the ligand

Review the hits as above.

When you are finished reviewing the hits, display the highest scoring hit of the second run by making it visible.

Select the Select BORDER Display button. The Select BORDER Display button is the last of the three display mode buttons on the upper right of the Visualizer.

Make l682 current. Move the hit into the l682 window by clicking anywhere within its display box (being careful not to click the structure itself) and dragging it into l682's display box.

Notice the alignment of the hit and the ligand L682. The hydrogen on the nitrogen of L682 is close to the ethyl carbon bonded to the phenyl group of the hit and the C-C bond of this ethyl group approximately aligns with the H-N bond of the L682 ligand. It is these bonds, the hydrogen and the methyl of the ethyl group that are replaced when these molecules are merged to form a ligand.

Delete the methyl group on the high-scoring hit that overlaps l682. This is done by selecting the methyl group and typing ctrl-delete. Similarly, delete the NH2 hydrogen (id=40) on l682.

Go to Build/3D-Sketcher... on the Visualizer. Now bond the two structures by selecting Connect (one of the last buttons on the bottom right) and then clicking the N on l682 and dragging the green dotted line over to the C on the hit.

A bond will form between the two structures.

10.   Tutorial complete

To review, you have used Ludi to add a new group of atoms to an existing inhibitor of the HIV protease enzyme. The modification suggested by Ludi adds new hydrophobic interactions between the inhibitor and the enzyme.

You can add other groups to other parts of the inhibitor now at your discretion.

To assure a fresh start for the next tutorial, reinitialize Cerius2. Select File/New Session... and click Confirm on the prompt that appears.

This has the effect of deleting all objects from the Model Window and reinitializing all of the parameters in Ludi.

Return to Tutorials contents.

Proceed to next tutorial.