Cerius²·Ludi

6.1       Tutorial lesson 1: de novo inhibitor design for trypsin

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Ludi can be used to design inhibitors de novo, requiring only a receptor structure and a coordinate in the active site.

In this tutorial you will use Ludi to search for fragments that can bind to trypsin, a proteolytic enzyme.

Tutorial

• Running Ludi in receptor-based mode: de novo ligand design.

• Loading Ludi hits.

• Reviewing hits.

• Loading interaction sites.

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 benzamidine.msi and then trypsin.msi.

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

In the Visualizer, select trypsin's diamond-shaped currency button and set benzamidine 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 the serine protease, trypsin (lower left), and a trypsin inhibitor, benzamidine (upper right). The heavy atom coordinates come from entry 3PTB in the Brookhaven Protein Data Bank.

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 corresponding fragment.

Note that the 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 de novo mode and the Link Library which is used when Ludi is run in link mode.

In this tutorial you will use a de novo library designed specifically for these tutorials.

Set the Library Type under De Novo Library to User. Find ludi_tut_std_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 Define Current Model as Receptor button to initialize trypsin as the receptor.

Leave Search Sphere Radius set to 5.00 Å and Maximum RMS to 0.30 Å.

This indicates that only trypsin atoms within 5 Å of the Search Sphere Center Coordinates are to be used in fitting fragments from the specified fragment library.

Now you will fill Search Sphere Center Coordinates with the coordinates of the C1 atom in benzamidine. If necessary, press the right mouse button to rotate benzamidine into view and the two rightmost mouse buttons to zoom in on the molecule. Next, click the C1 atom of benzamidine. This is the only phenyl carbon without a hydrogen. To make sure you have selected the correct atom, look at the report in the textport. It should read: Model(name=benzamidine):Chain(name=ABEN):Residue(type=BEN 1):Atom(name=C1) Now click Define Center From Selected Atoms.

When you are designing inhibitors de novo, you may wish to fill in the Search Sphere Center Coordinates parameter by picking an atom that is part of the active site of the protein. Or, you could create a small molecule (e.g., with a command like Build/3D-Sketcher...) and place it in the active site.

You can then pick atoms in the small molecule to fill in the Search Sphere Center Coordinates by clicking the Define Center From Selected Atoms button. The small molecule can be positioned wherever you like in the active site. As long as the Link Site parameter is unchecked, Ludi ignores all molecules except the receptor so the small molecule's presence does not perturb Ludi.

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

Open the Job Control panel by clicking 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 3 hits were found. If you get an error message that says the logfile does not exist, you've clicked the Monitor Logfile button too soon. Wait a few moments and try again.

6.   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 three 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 hits are loaded by score from highest to lowest. If the scoring information is not available, the hits are loaded in the order in which they were found in the Ludi library.

7.   Reviewing the results of the Ludi Run

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

Select all trypsin 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 benzamidine 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 benzamidine by placing your cursor next to it and dragging a box around it. Now, turn benzamidine 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 benzamidine in trypsin. If not, try another color.

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

If you wish to see how the hits fit in the interaction site, make sure that trypsin's visibility button is on and benzamidine's is off. If you wish to view the hits in comparison with benzamidine, deselect trypsin's visibility button and click benzamidine's on.

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

8.   Loading the interaction sites

To load the interaction sites use the Ludi Load control panel. Uncheck Hits and check Interaction Sites.

Click LOAD to load the interaction sites. Click the visibility button of the new model.

The interaction sites are now displayed in the graphics area. The vectors that are half red and half grey are hydrogen acceptor sites, with red for the acceptor and grey for the acceptor antecedent. The blue and white vectors are hydrogen donor sites with white for the hydrogen and blue for the heavy atom. The hydrophobic sites are at the vertices of the lines that are entirely grey.

For a description of how Ludi generates interaction sites and tries to fit fragments to them please refer to the Theory section.

9.   Tutorial complete.

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.

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