Cerius²·Ludi

6.4       Tutorial lesson 4: active analog mode

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Ludi can be used to design inhibitors when the structure of the receptor is not known but a set of ligands with a common biophore is known.

In this tutorial you will use Ludi to design a novel ligand for the HMG CoA system.

Tutorial

• Performing an analogs-based Ludi run.

• Running Ludi in link-specified mode.

• Loading Ludi hits.

• Reviewing hits.

• Loading interaction sites and analogs shell.

To start the tutorial select the File/Load Model... command, set File Format to MACCS, go to the Cerius2-Resources/LUDI directory and double click hmg_analogs.sd. In the Visualizer, select all of the models' visibility buttons.

2.   Examining the aligned active analogs

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 all of the molecules' visibility buttons are selected.

The molecules displayed on the screen are inhibitors of HMG CoA Reductase, a rate-limiting enzyme in the biosynthesis of cholesterol. HMG CoA Reductase is a target for efforts to design drugs to treat high blood cholesterol.

Before Ludi can be used to design a new inhibitor, the active analogs must be superimposed according to the biophore for the system.

The biophore for this example includes two hydroxyl groups, a carboxylic acid group and a p-fluorophenyl ring. This biophore will be the starting point for designing a new HMG CoA Reductase inhibitor. Ludi will suggest fragments that connect the portions of the biophore in novel ways.

3.   Viewing the Biophore

A scaffold containing the biophore is provided.

To load the biophore, go to the File/Load Model... command, set File Format to MACCS, go to the Cerius2-Resources/LUDI directory and double click hmg_biophore.mol. In the Visualizer, select all of the models' visibility buttons.

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.

4.   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 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 these 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.

5.   Setting the Ludi preferences

Go to the ANALOGS 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.

6.   Running the Ludi job

You are now ready to perform the Ludi run.

On the ANALOGS BASED DESIGN card, click Find Hits.

The Ludi Receptor Based Design panel will appear.

In the Visualizer's Models panel, select the three HMG_ANALOGS_* models by clicking to the left of the ID number of one and then holding down the shift key and clicking to the left of the other two model IDs to add them to the selection. Click the Defined Selected Model as Active Analogs button to define the set of active analogs.

Set the Search Sphere Radius set to 99 Angstroms.

This indicates that the whole volume occupied by the active analogs is considered for fragment fitting.

Set the Maximum RMS parameter to 0.4 Angstroms.

To fill the Search Sphere Center Coordinates select the terminal ethylene carbon (id=13) of hmg_biophore. Now click Define Center From Selected Atoms.

Now set the linkage sites. Select the hydrogen that is pointing toward the phenyl group, and then hold down the shift key and select the closest phenyl hydrogen.

Check Link Sites and click Define Links from Selected Model to establish that each hit must be aligned with a specified link site on the ligand.

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

Note that there is no setting for the Scoring Function on the Ludi Active Analog Based Design card. This is because only the Energy_Estimate scoring functions are suitable for use with receptor-based runs. For an active analog mode run, the Hbond_Lipo scoring function is used.

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 eight 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.

7.   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 ANALOGS BASED DESIGN card. When the Ludi Load panel appears, turn off Generate Hydrogen Bonds. 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.

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.

8.   Reviewing the results of the Ludi run

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 hmg_biophore's visibility button is selected.

Now view each hit in turn by making it the current model (make each model current by selecting its diamond shaped currency button in the Visualizer).

The fragment Model7 is a phenyl bridge. This bridge is used in an HMG CoA Reductase inhibitor designed at Merck.

Model9 is an ethylene bridge. This bridge is used in an HMG CoA Reductase inhibitor designed at Bristol-Myers Squibb.

When you are finished reviewing, make Model7 visible by clicking its visibility button, and make hmg_biophore current by selecting its diamond shaped currency button in the Visualizer.

9.   Bonding the ludi fragment to the biophore

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.

Click anywhere within Model7's display box (being careful not to click the structure itself) and drag it into hmg_biophore's display box.

Delete the two methyl groups on Model7 and the two hmg_biophore hydrogens that are overlapped by the methyls. This is done by selecting each atom and typing ctrl-delete.

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 and dragging the green dotted line from one atom to the next. You should connect Model(name=hmg_biophore):Chain(name=A071):residue(type C781):Atom(name=C4) with atom id=13 and atom id=4 with Model(name=hmg_biophore):Chain(name=A071):residue(type C781):Atom(name=C5).

10.   Loading the interaction sites and analogs shell

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

11.   Viewing the Ligand Inside the Pseudo Protein

Make Model14 current by selecting its diamond shaped currency button in the Visualizer.

Select all of Model14's atoms by placing your cursor next to it and dragging a box around it.

Change the display by selecting BALL from the Atom Display Style popup in the upper left hand corner of the Visualizer.

Select View/Colors... on the Visualizer and set the Pen popup to BLUE.

12.   Coloring the Ligand

Make hmg_biophore current by selecting its diamond shaped currency button in the Visualizer.

Select all of hmg_biophore's atoms by placing your cursor next to it and dragging a box around it.

Select View/Colors... on the Visualizer and set the Pen popup to MAGENTA.

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.

In the Visualizer, go to View/Graphics Z-Clipping..., check Apply Z-Clipping and set Front Z-Clip Plane to 0.38 on the Z-Clipping control panel.

Now go to View/Dials... and set Magnification to 1.25 on the Viewing Dials control panel.

The yellow, grey and red lines represent the Ludi interaction sites. The lines that are half grey and half red are hydrogen bond accepting sites with grey for the acceptor antecedent and red for the hydrogen acceptor. The vertices of the all-grey lines are hydrophobic interaction sites and the yellow lines are link sites.

13.   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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