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(Molecular Fingerprinting and Docking of Compounds to the ADA active site)
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==Molecular Fingerprinting and Docking of Compounds to the ADA active site==
+
==Molecular Fingerprinting of Compounds to the ADA active site==
 +
* The objective for this laboratory period is obtain the molecular fingerprint of aspirin and dock compounds to the binding pocket of adenosine deaminase (ADA).
 +
 
  
 
A. Opening Maestro  
 
A. Opening Maestro  
Line 29: Line 31:
 
* Minimization analyzes the molecule using Classical Physics.
 
* Minimization analyzes the molecule using Classical Physics.
 
* A dialog box appears with the force field set on OPLS 2005.
 
* A dialog box appears with the force field set on OPLS 2005.
 +
** Force field is a set of rules containing how atom types are connected angle and torsional angle
 +
** gives the constant measure of force
 +
** The Constraints tab contain NOE, NMR, indicating the closeness of atoms and information about the radius
 
* Then click the Minimization tab. On the maximum cycles, this was changed to 10,000. The other standard parameters such as gradient criterion (0.01) and energy change criterion (1e-07) were kept. The pH was changed to the desired pH level of 7.4.
 
* Then click the Minimization tab. On the maximum cycles, this was changed to 10,000. The other standard parameters such as gradient criterion (0.01) and energy change criterion (1e-07) were kept. The pH was changed to the desired pH level of 7.4.
 
* Knowing there are amino acids present, by changing the pH of the parameters, the structure must also be converted to its alkoxide form at pH 7.4. As a result, through atom types O3 was changed to OM.
 
* Knowing there are amino acids present, by changing the pH of the parameters, the structure must also be converted to its alkoxide form at pH 7.4. As a result, through atom types O3 was changed to OM.
 
* After all the parameters were set for minimization, the host zorro was chosen and the minimization was started.
 
* After all the parameters were set for minimization, the host zorro was chosen and the minimization was started.
* Double-click the finished minimization on the monitor jobs (from applications option)
+
* Double-click the finished minimization on the monitor jobs (from applications option), then label the partial charges of the molecule.
 +
* Save the project entry.
 +
 
 +
 
 +
C. Calculation of Fingerprint
 +
 
 +
* Opened Canvas from the Finder by typing $Canvas
 +
* Created a new project and named it as Aspirin_fingerprint.
 +
* Imported the energy minimized aspirin saved earlier from maestro.
 +
* Under applications option, click binary fingerprints > molprint2D
 +
* Click on the imported molecule and incorporate.
 +
* Export the molecule by saving it with an extension of .fp
 +
* A dialog box will appear, choose remove all properties and click ok.
 +
* Close the project.
 +
 
 +
 
 +
D. Screen Fingerprint
 +
 
 +
* After closing the previous aspirin project, open the zinc database.
 +
* Imported the aspirin.fp molecule and allow duplicate mappings.
 +
* Go to the Applications > similar/distance screen > select aspirin.
 +
** Tanimoto similarity
 +
* Selected the fingerprint column and choose the fingerprint (aspirin)
 +
* Screened the molecules and then incorporate ~ 15,001
 +
* Clicked the fingerprint screen column and sort in descending order to show molecules closest to aspirin.
 +
* On the panel, select rows 1-15,001
 +
* Saved the selection by exporting the project as aspirin_15001.mae along with its properties and click OK.
 +
** A noticeable common feature among the imported database is the benzene ring.
 +
* Closed Canvas
 +
 
 +
 
 +
E. Docking Preparation
 +
[[Image:Prepwizard2.png|thumb|right|Protein Preparation Wizard_Import and Process Tab]]
 +
* Opened the protein databank (PDB) website, www.rcsb.org, and acquired two ADA Bovine structures. The two following structures were chosen by desirable resolutions:
 +
**1KRM (resolution 2.5 angstroms)
 +
**2Z7G (resolution 2.52 angstroms)
 +
* The pdb.txt of these structures were downloaded.
 +
* On Maestro, opened the aspirin.prj and imported the downloaded structures of ADA.
 +
* The structures of ADA were protonated according to the pH of 7.4. This was executed from:
 +
** Workflows > Protein preparation Wizard
 +
** On the Import and Process Tab, add H<sup>+</sup> (see image on the right side of the protein preparation wizard dialog box)
 +
** Uncheck the delete waters on the dialog box and click preprocess.
 +
** Moving to the Refine Tab, changed the default pH of 7.0 to 7.4 as shown below.
 +
** Then click Optimize
 +
 
 +
[[Image:Refine75.png|center]]
 +
 
 +
 
 +
F. Superimposition of Proteins
 +
 
 +
* After Optimization, navigated through Tools > Protein Structure Alignment
 +
** All > align
 +
** From the Undisplay icon, remove waters for both structures
 +
* Added ribbons for all residues to view the entire structure
 +
* Upon scanning the structure, subtracted all portions leaving only the ligand on display at 5 Angstroms.
 +
* Compared both structures by superimposing them.
 +
* Verified structure 1KRM has a better resolution and removed 2Z7G from the project table.
 +
* Showed ribbons for 1KRM and colored element entry carbons
 +
 
 +
 
 +
G. Docking
 +
* Duplicated the 1KRM H-minimized and deleted water molecules by manually clicking on each.
 +
** Removed the water molecules to remove the rigidity of the protein. This also interferes with the docking of compounds by taking up space.
 +
* Changed ribbon color by residue position
 +
* Made a grid of the docking region by:
 +
** Application > Glide > Receptor Grid Generation
 +
** Select the ligand
 +
** Click site tab > center on ligand > change dock ligands to 15 Angstroms
 +
** Click Start
 +
* Changed the grid name and specified the host: zorro; click OK
 +
 
  
 +
* Seeing the docking is done from the Monitor Jobs window:
 +
** Applications > Glide > Ligand Docking
 +
** Browse for the .zip grid previously created
 +
** Verify host: zorro and click start
 +
* Imported the glide.pv file
 +
* Excluded the protein structure leaving only the ligand
 +
* Superimposed the raw, crystallized PDB ADA ligand structure with the docked ligand
 +
* Imported flavonoids to compare with the docked ligand by superimposition
 +
* Database screening was initiated by going to Applications > Glide > Ligand Docking
 +
* Selected the entry with 15,000 compounds from the .pv file
 +
* Selected zorro host and clicked start
  
 +
==Notes==
  
 +
* The database chosen were compounds that did not cross the Blood Brain Barrier.
  
 
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Revision as of 08:48, 8 February 2013

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Molecular Fingerprinting of Compounds to the ADA active site

  • The objective for this laboratory period is obtain the molecular fingerprint of aspirin and dock compounds to the binding pocket of adenosine deaminase (ADA).


A. Opening Maestro

  • From the assigned computer, a new terminal was created from either of the following steps:

1. Finder > Services > New terminal at folder

-or-

2. press the provided shortcut key F5

  • Then enter $maestro to open the suite.
  • Draw the molecule on the workspace area. Modifications can be made through the Edit panel > build > fragments > atom properties. Sponge the drawn molecule for clarity.
  • To save the molecule, create this entry on the project table -or- from the workspace option > project entry > name and create the project and then save.
  • The next step is to put the molecule under energy minimization to find a stable energetically conformation.


B. Energy Minimization

  • To initiate energy minimization, go to the Applications option > Impact > Minimization.
  • Minimization analyzes the molecule using Classical Physics.
  • A dialog box appears with the force field set on OPLS 2005.
    • Force field is a set of rules containing how atom types are connected angle and torsional angle
    • gives the constant measure of force
    • The Constraints tab contain NOE, NMR, indicating the closeness of atoms and information about the radius
  • Then click the Minimization tab. On the maximum cycles, this was changed to 10,000. The other standard parameters such as gradient criterion (0.01) and energy change criterion (1e-07) were kept. The pH was changed to the desired pH level of 7.4.
  • Knowing there are amino acids present, by changing the pH of the parameters, the structure must also be converted to its alkoxide form at pH 7.4. As a result, through atom types O3 was changed to OM.
  • After all the parameters were set for minimization, the host zorro was chosen and the minimization was started.
  • Double-click the finished minimization on the monitor jobs (from applications option), then label the partial charges of the molecule.
  • Save the project entry.


C. Calculation of Fingerprint

  • Opened Canvas from the Finder by typing $Canvas
  • Created a new project and named it as Aspirin_fingerprint.
  • Imported the energy minimized aspirin saved earlier from maestro.
  • Under applications option, click binary fingerprints > molprint2D
  • Click on the imported molecule and incorporate.
  • Export the molecule by saving it with an extension of .fp
  • A dialog box will appear, choose remove all properties and click ok.
  • Close the project.


D. Screen Fingerprint

  • After closing the previous aspirin project, open the zinc database.
  • Imported the aspirin.fp molecule and allow duplicate mappings.
  • Go to the Applications > similar/distance screen > select aspirin.
    • Tanimoto similarity
  • Selected the fingerprint column and choose the fingerprint (aspirin)
  • Screened the molecules and then incorporate ~ 15,001
  • Clicked the fingerprint screen column and sort in descending order to show molecules closest to aspirin.
  • On the panel, select rows 1-15,001
  • Saved the selection by exporting the project as aspirin_15001.mae along with its properties and click OK.
    • A noticeable common feature among the imported database is the benzene ring.
  • Closed Canvas


E. Docking Preparation

Protein Preparation Wizard_Import and Process Tab
  • Opened the protein databank (PDB) website, www.rcsb.org, and acquired two ADA Bovine structures. The two following structures were chosen by desirable resolutions:
    • 1KRM (resolution 2.5 angstroms)
    • 2Z7G (resolution 2.52 angstroms)
  • The pdb.txt of these structures were downloaded.
  • On Maestro, opened the aspirin.prj and imported the downloaded structures of ADA.
  • The structures of ADA were protonated according to the pH of 7.4. This was executed from:
    • Workflows > Protein preparation Wizard
    • On the Import and Process Tab, add H+ (see image on the right side of the protein preparation wizard dialog box)
    • Uncheck the delete waters on the dialog box and click preprocess.
    • Moving to the Refine Tab, changed the default pH of 7.0 to 7.4 as shown below.
    • Then click Optimize
Refine75.png


F. Superimposition of Proteins

  • After Optimization, navigated through Tools > Protein Structure Alignment
    • All > align
    • From the Undisplay icon, remove waters for both structures
  • Added ribbons for all residues to view the entire structure
  • Upon scanning the structure, subtracted all portions leaving only the ligand on display at 5 Angstroms.
  • Compared both structures by superimposing them.
  • Verified structure 1KRM has a better resolution and removed 2Z7G from the project table.
  • Showed ribbons for 1KRM and colored element entry carbons


G. Docking

  • Duplicated the 1KRM H-minimized and deleted water molecules by manually clicking on each.
    • Removed the water molecules to remove the rigidity of the protein. This also interferes with the docking of compounds by taking up space.
  • Changed ribbon color by residue position
  • Made a grid of the docking region by:
    • Application > Glide > Receptor Grid Generation
    • Select the ligand
    • Click site tab > center on ligand > change dock ligands to 15 Angstroms
    • Click Start
  • Changed the grid name and specified the host: zorro; click OK


  • Seeing the docking is done from the Monitor Jobs window:
    • Applications > Glide > Ligand Docking
    • Browse for the .zip grid previously created
    • Verify host: zorro and click start
  • Imported the glide.pv file
  • Excluded the protein structure leaving only the ligand
  • Superimposed the raw, crystallized PDB ADA ligand structure with the docked ligand
  • Imported flavonoids to compare with the docked ligand by superimposition
  • Database screening was initiated by going to Applications > Glide > Ligand Docking
  • Selected the entry with 15,000 compounds from the .pv file
  • Selected zorro host and clicked start

Notes

  • The database chosen were compounds that did not cross the Blood Brain Barrier.