User:Mary Mendoza/Notebook/CHEM572 Exp. Biological Chemistry II/2013/01/30

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

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.