Molecular Fingerprinting of Compounds to the ADA active site
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
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.
- 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
- 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
- The database chosen were compounds that did not cross the Blood Brain Barrier.