Cerius²·Ludi

5       General methodology

Ludi extracts from a library of molecular fragments those fragments that have attributes characteristic of ligands for a particular protein. The Theory chapter, describes the theory behind Ludi. This chapter describes how to use Ludi.

The Cerius² interface to Ludi is divided into two different modes of operation.

One mode, RECEPTOR BASED DESIGN, is used when the structure of the protein is known. The second mode, ANALOGS BASED DESIGN, is used when the structure of the protein is not known but analogs are known (see Generating ligand candidates based on active analogs). Active analogs are molecules that contain the same biophore.

Generating novel ligand candidates for a protein of known structure

Ludi is used in a variety of ways to design molecules that are potential ligands:

• Ludi can find one or more molecular fragments and suggest how they could be bonded together (using Cerius²) to form a potential ligand.

• Ludi can find fragments which you then bond together in Cerius² using bridge fragments of your own design.

• Ludi can modify a known ligand by replacing certain portions of it with Ludi-suggested fragments. The portions you replace can be at the ends of the ligand or in the interior.

• Ludi can suggest fragments to add to a known ligand that may enhance its activity.

1.   Load the model protein into Cerius²

Use File/Load Model... to load the protein.

The Cerius² module Active Site Viewer is helpful. See The Active Site Viewer chapter.

If required, use the Ludi Runtime Parameters panel to adjust the parameters. Adjusting these parameters is often not necessary since the default values provided can be used in many cases.

• If you have created and want to use an alternate Ludi library, first open the Ludi LIBRARY card and click Library Spec to bring up the Ludi Library Specification panel.

• Then under De Novo Library, set the Library Type parameter to User.

• Specify the alternate fragment topology file by highlighting it in the file browser and clicking the SELECT button.

Ludi is executed by clicking Find Hits and then clicking the Find Ludi hits button.

Note that the Find Hits button is available on both the RECEPTOR and ANALOGS BASED DESIGN cards.

In this case, since you know that protein's structure, you should run Ludi in the Receptor mode.

• Select the RECEPTOR BASED DESIGN card from the Ludi deck of cards and click Find Hits to bring up the Ludi Receptor Based Design panel.

• Make sure that your protein is the current model (i.e., make sure that its diamond-shaped currency button is selected in the Visualizer) and click the Define Current Model as Receptor: button.

• The Search Sphere Radius parameter specifies the size of the sphere in which Ludi is to fit appropriate fragments. In addition, Ludi looks within this sphere for receptor atoms. The default value is 5.00 Å.

• The Search Sphere Center Coordinates parameter specifies the center of the sphere within which Ludi will conduct its search.

There are three ways to fill the Search Sphere Center Coordinates. You may type the coordinates directly, specify Selected atom(s), or instruct Ludi to calculate the center of search from the atoms in the Active Site definition that is created with the Active Site Viewer.

Once you have made your selection, click the Define button. The coordinates will appear in the display (X, Y, Z) and are marked in the graphics window by a yellow crosshatch. The crosshatch is sometimes difficult to find, so you should click the two right-most mouse buttons and drag your mouse up to zoom in on the model.

• The Define No Links button allows you to specify whether Ludi is run in link mode. The default setting of No Links indicates that Ludi should not be run in link mode; i.e., Ludi does not need to take into account any other molecules in the active site.

In this case, leave this parameter set to No Links since this is the first iteration and you are not adding to a partially completed candidate ligand.

• If you wish to require that fitted fragments interact with certain target atoms on the receptor model, first select the desired atoms in the Cerius² Model window.

To highlight the target atoms, use the mouse to draw a box around them or select them one by one with the mouse while holding down the <shift> key.

Once all of the target atoms have been selected, click the Define Selected Atoms as Target Atoms button.

• Finally, click Find Ludi hits to begin the Ludi job.

While the Ludi job is running you can check its progress using the Job Control feature.

Click Job Control to bring up the Ludi Job Control panel and use the Monitor Logfile or Completion Status buttons to monitor the job status.

When the Ludi run completes, load the results into Cerius².

The Ludi Load panel is used to load the results of a Ludi run into Cerius².

• First, click Load on the RECEPTOR BASED DESIGN card to bring up the Ludi Load control panel.

• Usually, you will want to load only the Hits, but you can load Interaction Sites as well. Analogs Shell applies only to analogs based runs.

• Use the file browser to select the .run file for the Ludi run.

• Note that Generate Hydrogen Bonds is on by default. You should click the Define Current Model as Receptor button to define the receptor model (remember to make sure that the correct protein is the current model, as above), if the label is not already set to your receptor model.

• When Load into Study Table is unchecked, the hits are loaded to the score table. When checked, the hits are loaded into a QSAR study table. The second option is unavailable if you lack a QSAR license.

• If you wish you can use Hit Selection to load a subset of the results.

• Now click the Load button to load the results of the Ludi run.

To view the way the fragments were fit, put the graphics windows in overlay mode, and make the receptor visible and all fragments invisible. Make each fragment current to view them one by one in the receptor.

After examining the fragments choose one of them to continue the building process. Choose a fragment that you think is promising, e.g., oriented well for continuing to "grow" the ligand candidate in the desired direction, one that can be synthesized in the laboratory, etc.

For the purposes of this description the chosen fragment is referred to as FRAG1.

• Adjust the Ludi parameters as needed, using the Ludi Runtime Parameters panel accessible by selecting Preferences on the RECEPTOR BASED DESIGN card.

• Change the Define No Links button to Single Links, Double Links, or Links depending on how you want to build the ligand candidate (refer to Figure 1).

For the purposes of this description, consider setting Define No Links to Single Links (since you want Ludi to find fragments to link with FRAG1).

• Select FRAG1 and click the Define Single Links from Selected Model button.

• Pick one of the atoms on FRAG1 to define the Search Sphere Center Coordinates, as above.

• When the Ludi run completes, load the latest results using the Ludi Load panel as above.

• Review the Ludi results (as above).

Note that each fragment is positioned in the way that Ludi considered best, however Ludi has not created a bond between the suggested fragment and the ligand candidate. Based on your criteria of growing direction, ability to synthesize, etc., choose one of the fragments to be the next piece of the ligand candidate.

For the purposes of this description, the fragment chosen is referred to as FRAG2.

• Merge and bond the fragments.

Put the graphics window in BORDER view. Make FRAG1 current and FRAG2 visible. Drag the FRAG2 window into the FRAG1 window to merge them into a single model. Use the 3D-Sketcher (under the Build pulldown in the Visualizer) to delete overlapping atoms and create a bond between the two fragments.

Iterate the following sequence of Ludi commands until you are satisfied with the ligand candidate.

• Modify the Ludi parameters as needed in the Ludi Runtime Parameters panel.

• Run the Ludi program specifying the linkage and Search Sphere Center Coordinates.

• Load the Ludi results into Cerius² using the Ludi Load panel.

• Review the results (as above) and choose the fragment you think is best.

• Merge and bond (as above) the chosen fragment to the partially completed ligand.

• Load in a protein and an inhibitor using File/Load Model... or another Cerius² command.

• Determine the part(s) of the inhibitor that you want to replace and remove them using Cerius² commands. For example, you could use Edit/Delete to remove selected atoms.

• Optionally, specify an alternate Ludi library.

If you have created an alternate Ludi library and want to use it, go to the Ludi LIBRARY card and click Library Spec. On the Ludi Library Specification panel, set the Library Type option (under Link Library) to User. Specify the alternate fragment topology file in the Library text field by picking it from the file browser and clicking SELECT.

• Execute the Find Ludi hits command with the linkage set to either Double Links or Links. Define Double Links indicates that you want Ludi to suggest fragments that can make two links to the ligand, this includes fragments that bridge the gap between pieces of the ligand. Define Links allows you to select the link sites for Ludi to use. Any hydrogen atom constitutes an allowable link site.

While the Ludi job is running you can check its progress using the Job Control feature, as above.

• Once the Ludi run has completed, examine the results of the run, as above and then choose a fragment to continue building the ligand candidate.

• Merge and bond (as above) the chosen fragment to the partially completed ligand.

• As in the other procedures, begin by loading in the protein and ligand and, optionally, specify an alternate library.

• Click Find Hits to open the Ludi Receptor Based Design panel. Set the linkage to Single Links, Double Links, or Links. For Define Links, select link atoms on the ligand. For Define Double or Single Links, select the ligand model and the link sites will be found automatically. Finally, set the Search Sphere Center Coordinates, as above. This can be done by picking a ligand atom that is in the area to which you want to add and then clicking the Define Center From Select Atoms button.

• When the Ludi run completes, load and review the results using the commands on the Ludi Load panel. Choose one of the fragments.

• Merge and bond (as above) the chosen fragment to the existing ligand.

The following steps outline the procedure for building a ligand candidate when you know the active ligand(s) but do not know the structure of the receptor.

Choose the analog(s). The set of analogs should reflect the biophore for which you want to build a ligand candidate.

1.   Load the model(s) into Cerius²

If you have more than one analog, superimpose them according to the biophore. The way the analogs are superimposed is critical to the quality of the results since Ludi constructs a hypothetical active site based on the superimposed analogs.

Select the analog models and click the Define Selected Models as Active Analog button to set the definition.

If required, use the Ludi Runtime Parameters panel to adjust the parameters. Adjusting these parameters is often not necessary since the default values provided can be used in many cases.

• If you have created and want to use an alternate Ludi library, first open the Ludi LIBRARY card and click Library Spec to bring up the Ludi Library Specification panel.

• On the Ludi Library Specification panel, set the Library Type option (under De Novo Library) to User in order to specify an alternate library.

• Specify the alternate fragment topology by highlighting it in the file browser and clicking the SELECT button.

Before running Ludi, the parameters should be set as follows:

• The Search Sphere Radius parameter specifies the size of the sphere in which Ludi is to fit appropriate fragments. The default value is 99.00 Å.

• The Search Sphere Center Coordinates parameter specifies the center of a sphere within which Ludi will conduct its search.

• There are two ways to fill the Search Sphere Center Coordinates. You may either select atom(s) and click Define Center from Selected Atoms or enter the coordinates directly.

• The Define No Links button allows you to specify whether Ludi is run in link mode. The default setting of No Links indicates that Ludi should not be run in link mode; i.e., Ludi does not need to take into account any other molecules in the active site.

In this case, leave this parameter set to No Links since this is the first iteration and you are not adding to a partially completed candidate ligand.

• Finally, click Find Ludi hits to begin the Ludi job.

While the Ludi job is running you can check its progress using the Job Control feature.

Click Job Control to bring up the Ludi Job Control panel and use the Monitor Logfile or Completion Status buttons to monitor the job status.

When the Ludi run completes, load the results into Cerius².

The Ludi Load panel is used to load the results of a Ludi run into Cerius².

• First, click Load on the ANALOGS BASED DESIGN card to bring up the Ludi Load control panel.

• Usually, you will want to load only the Hits, but you can load Interaction Sites and the Analogs Shell as well.

• Use the file browser to select the .run file for the Ludi run.

• Note that Generate Hydrogen Bonds is on by default. You should turn it off for analog based runs.

• When Load into Study Table is unchecked, the hits are loaded to the browser. When checked, the hits are loaded into a QSAR study table. The second option is unavailable if you lack a QSAR license.

• If you wish you can use Hit Selection to load a subset of the run results.

• Now click Load button to load the results of the Ludi run.

Review the Ludi results (as above).

• To continue building the ligand follow the directions in steps 9. and 10. in Generating a ligand candidate de novo above.

For runs in which fragments are being fit at a metal ion interaction site, the Electrostatic Check parameter on the Ludi Runtime Parameters control panel must be unchecked.

Table 7 lists several parameters contained on the Ludi Runtime Parameters, Ludi Active Analog Based Design and Ludi Receptor Based Design control panels that can be modified if you experience the following problems:

• Ludi returns too few or no fragments.

• Ludi returns too many fragments to consider.

• Ludi runs very slowly.

  Table 7 . Trouble-shooting Ludi calculations

  Parameter Name	Adjustment or Setting	Control paneld	Recommended values
  Too few fragments suggested by Ludi
  Search Sphere Radius	Increase b	Design	4Å - 8Å
  Maximum RMS	Increase	Design	0.3 - 0.5 or 0.4 - 0.6 Å a
  Preselect	Increase b	Parameters	1.5 - 2.5
  Density of Lipophilic Sitese	Increase b	Parameters	10 - 25
  Density of Polar Sitese	Increase b	Parameters	15 - 25
  Aliphatic_Aromatic	checked	Parameters	n/a
  Reject Bifurcatede	checked	Parameters	n/a
  No Unpaired Polare	checked	Parameters	n/a
  Minimum Surfacee	Decrease	Parameters	n/a
  Maximum Unfilled Cavitye	Decrease	Parameters	0
  Too many fragments suggested by Ludi or Ludi runs too slowly
  Search Sphere Radius	Decrease	Design	4Å - 8Å
  Maximum RMS	Decrease	Design	0.3 - 0.5 or 0.4 - 0.6 Å a
  Define Links	Links c	Design	n/a
  Max Alignment Angle	Decrease c	Design	12° - 14°
  Preselect	Decrease	Parameters	1.5 - 2.5
  Density of Lipophilic Sitese	Decrease	Parameters	10 - 25
  Density of Polar Sitese	Decrease	Parameters	15 - 25
  Aliphatic_Aromatic	checked	Parameters	n/a
  Reject Bifurcatede	checked	Parameters	n/a
  No Unpaired Polare	checked	Parameters	n/a
  Minimum Surfacee	Increase	Parameters	n/a
  Maximum Unfilled Cavitye	Increase	Parameters	0
  a This parameter's recommended value depends on the value of the Define Linkage parameter. Lower values for No Links, and higher values for Single Links, Double Links, or Links.   b Increasing the values of these parameters slows the Ludi run.   c These parameters are applicable in link mode only.   d The Ludi Runtime Parameters and Design cards are available on the RECEPTOR BASED DESIGN and ANALOGS BASED DESIGN cards in the Ludi deck of cards.   e These parameters are applicable in receptor based mode only.

Ludi fragment bond orders

For new libraries, files can be written in either PDB or MOL file format. If MOL file format is used, then bond order information is retained. New libraries may be created using the Ludi Add Library Entry card (accessible via the Add Entry button on the Ludi LIBRARY card) or the UNIX-level command Ludi_Genlib.

The fragments in the default Ludi fragment libraries are stored in MDL Mol format.