Cerius²·Ludi

6.3       Tutorial lesson 3: targeted mode

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The targeted mode in Ludi allows you to specify an atom or atoms with which every hit must interact. This is particularly useful when your receptor is a metalloenzyme and the inhibitor must complex the metal.

In this tutorial you will use the Ludi targeted mode to search for fragments that bind to the zinc atom in carbonic anhydrase.

Tutorial

• Running Ludi in Targeted Mode.

• Loading Ludi hits.

• Reviewing hits.

• Setting run time parameters when fitting to metals.

Click carbonic_anhydrase's visibility button on and 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.

2.   Viewing the ligand in the receptor site

The molecules displayed on the screen are carbonic anhydrase and the inhibitor, 5-acetamido-1,3,4-thiadiazole-2-sulfonamide (AZM).

The heavy atom coordinates come from entry 1azm in the Brookhaven Protein Data Bank. The waters have been removed and hydrogens added.

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 standard, de novo library designed specifically for these tutorials.

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

4.   Setting the Ludi preferences

Go to the RECEPTOR BASED DESIGN card and select Preferences.

The Ludi Runtime Parameters panel will appear.

When you are fitting fragments to a metal, you must turn off the Electrostatic Check parameter. Otherwise, for this 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 off 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. First you will do the run without specifying any targets on the receptor.

On the RECEPTOR BASED DESIGN card, click Find Hits.

The Ludi Receptor Based Design panel will appear.

Select carbonic anhydrase by clicking to the left of its ID number in the Models panel in the Visualizer. Click the Defined Selected Model as Receptor button to initialize carbonic_anhydrase as the receptor.

Leave Search Sphere Radius set to 5.00 Angstroms.

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

Fill Search Sphere Center Coordinates with the coordinates of the S1 atom in AZM. To do this, make sure AZM is the current model by clicking its (diamond-shaped) currency button in the Visualizer. Then turn off carbonic anhydrase's (square) visibility button. AZM should now appear alone on the screen. If necessary, press the right mouse button to rotate AZM into view and the two rightmost mouse buttons to zoom in on the molecule. Next, click the S1 atom of AZM. To make sure you have selected the correct atom, look at the report in the textport. It should read: Model(name=azm):Atom(id=12) 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 Link Sites has been checked, Ludi ignores all molecules except the receptor so the small molecule's presence does not perturb Ludi.

To begin the Ludi calculation, click the 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 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.

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 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 hits are loaded in 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

Turn on carbonic_anhydrase's visibility button and turn off all other visibility buttons.

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

Some but not all of the Ludi fragments are interacting with the zinc atom.

8.   Defining receptor target atoms

In the next Ludi run, you will specify that all hits must interact with the zinc.

Make cabonic_anhydrase the current model and turn off AZM visibility. Select the purple zinc atom. The following should appear in the textport: Modle(name=carbonic_anhydrase):Chain(name=BCA):Residue(type=ZN Z61):Atom(name=ZN) Now click Define Selected Atoms as Target Atoms.

To begin the Ludi calculation, click the 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 three 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.

9.   Reviewing the Ludi hits

Load and review the results as above.

Notice that all three hits now interact with the zinc atom.

10.   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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