Structure-based Virtual Screening for Exploring Potential

Drugs from Database: A Practice of Computer-Aided (assisted)

Drug Design
Written by Siying Cen, Vahab Rajaei and Dr. Mary E. Peek
School of Chemistry and Biochemistry | Georgia Institute of Technology |Fall 2020

Structure-based drug design (SBDD) and ligand-based drug design (LBDD) are the two general
types of computer-aided drug design (CADD) approaches. SBDD methods involve the analysis
of structural information for macromolecular targets, typically of proteins or RNA, to identify
key sites and interactions that are important for their respective biological functions. Many types
of computational algorithms have been developed to sort compounds from chemical databases
by predicting the strength of interaction between ligands and targets. AutoDock Vina is an
automated docking algorithm that efficiently predicts how ligands bind to a target with a known
3D structure. PyRx is a drug discovery program that coordinates the action of several
computational tools including AutoDock Vina (for docking), AutoDock Tools (for file generation),
and Open Babel (for importing structure data format [SDF] files and energy minimization),
among other tools. SDF file format is the standard for ligand libraries in the pharmaceutical industry.
Molecular visualization tools such as PyMOL, Rasmol, and PMV facilitate viewing and analysis
of PyRx results.

In this experiment, students will dock two drug molecules within a chosen Anatomical
Therapeutic Chemical (ATC) class into a biological target to identify the best therapeutic within
the ATC class. The best therapeutic will have the tightest binding affinity and most plausible
structure-function impact.

Learning Goals
• Conduct docking experiments in docking software like PyRx or AutoDock Vina
• Compute Kd from Gibbs’ free energy of binding data provided by PyRx
• Visualize the ligand-protein results from PyRx and identify key interactions that
stabilize binding
• Correlate the impact of ligands on the structure and function of protein targets

Materials
Software can be accessed using VLab. For instructions on accessing VLab, see the protocol addendum
• ChEMBL compound library https://www.ebi.ac.uk/chembl/
• PyRx (containing AutoDock Vina and AutoDock Tools)
• PyMOL
• Chem3D (ChemDraw3D)

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 Experimental Procedures
Protein File Preparation

1. Choose one interesting biological target (i.e. protein) to study from column “PDB_1” or
“PDB_2” in Table 1. Write its PDB ID Code in your ELN.

Table 1. ATC Classification Level 2 Protein Targets with their Corresponding PDB ID Codes
ATC_class2 target_1 PDB_1 target_2 PDB_2
Isocitrate dehydrogenase
A07 1J1W Thioredoxin reductase 1 2PUK
[NADP] cytoplasmic
A10 Sodium/glucose cotransporter 3DH4 Cytochrome P450 3A4 2V0M
C01 Microtubule-associated protein 5N74 Coagulation factor X 4BTT
D01 Dopamine D2 receptor 6CM4 Adenosine A2a receptor 3EML
ATPase family AAA domain-
G01 DNA polymerase iota 3EPG 5A5R
containing protein 5
Tyrosyl-DNA
G03 1QZQ Dopamine D2 receptor 6CM4
phosphodiesterase
ATPase family AAA domain-
J01 6YB4 Geminin 4BRY
containing protein 5
Tyrosyl-DNA
J05 1QZQ Cytochrome P450 3A4 2V0M
phosphodiesterase
Tyrosyl-DNA
L01 1QZQ Cytochrome P450 3A4 2V0M
phosphodiesterase
M01 Dopamine D2 receptor 6CM4 Aldehyde dehydrogenase 1A1 5AC2
N02 Thioredoxin reductase 1 2PUK Neuropeptide S receptor 5ZBH
Bromodomain adjacent to zinc
N05 DNA polymerase iota 3EPG 2E7O
finger domain protein 2B
Glucagon-like peptide 1
N06 3C59 Neuropeptide S receptor 5ZBH
receptor
P01 Bile salt export pump 6LR0 Cytochrome P450 3A4 2V0M
Tyrosyl-DNA
R01 1QZQ Geminin 4BRY
phosphodiesterase

R03 Dopamine D2 receptor 6CM4 Tyrosyl-DNA phosphodiesterase 1QZQ

Muscarinic acetylcholine
R06 5CXV Lysosomal alpha-glucosidase 5NN6
receptor M1
Tyrosyl-DNA
S01 1QZQ Aldehyde dehydrogenase 1A1 5AC2
phosphodiesterase

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2. Download the PDB file from the Protein Data Bank < https://www.rcsb.org/ >
a. Enter the PDB ID Code into the search window
b. In the Download Files pulldown (Figure 1), select PDB format [not PDB Format
(gz)]

Figure 1. Screenshot showing Download File pulldown in the red box for obtaining a PDB file

3. Open a text editor such as TextEdit, WordPad, or Notepad on your computer and open
the PDB file using the text editor:
a. Click File à Open à Navigate to the Desktop
b. Change file type to “Any file type (*.*)” to be able to select the PDB file.

4. Find the coordinates for the ligands and water in the PDB file. They will usually be at
the bottom of the file with the line label as HETERO or HETATM for heteroatom
designation.
a. Select the ligand and water atom lines and delete them from the file.
b. Delete any lines specifying the connectivity between these heteroatoms in your
new file as these are incorrect.
c. Save as a newly named PDB format by clicking File à Save As... Save file with a
new name (e.g. newproteinname.pdb).

5. Import the target PDB file into PyRx and convert it to PDBQT file format:
a. In PyRx, click the Molecules tab (Figure 2). Then use File à Load Molecule to
upload target.pdb.

b. Once you see the name of your modified PDB file in the Molecules tab, right
click it and select Autodock à Make Macromolecule to convert it to a .pdbqt
file. This procedure will automatically conduct several additional edits
including:

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 i. Adding polar hydrogens
ii. Removing extraneous water molecules
iii. Adding partial charges
iv. Merges nonpolar hydrogens

Figure 2. Illustration of a protein target that has been loaded into PyRx for conversion to PDBQT format

Ligand File Preparation

6. Go to the ChEMBL compound library < https://www.ebi.ac.uk/chembl/ >

7. Scroll down the home page to find and click the “Browse all ChEMBL” icon

8. Scroll down the Compounds page to see the ATC Classification Level 2 filter in the left
panel (Figure 3). Select the appropriate filter from Table 1 that correlates to the target
that you selected.

Figure 3. Excerpt of ATC Classification Level 2

Pharmaceuticals with the A07 group (Antidiarrheals,
Intestinal Antiinflammatory / Antiinfective Agents)
highlighted

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9. Search for two interesting drugs within your chosen ATC Classification Level 2 class to
study and draw them in Chem3D:
a. Click on the ChEMBL ID code to open each drug’s Compound Report Card
b. Scroll down to the REPRESENTATIONS area to copy the Canonical SMILES
representation information
c. Open Chem3D (i.e. ChemDraw3D)
d. Select Edit à Paste Special à SMILES
e. The structure of the molecule that you chose should be visible

10. Produce and save separate Chem3D files for each of your selected drugs. For each drug
(ligand) file, conduct an energy minimization. Choose either one of the methods below
to do so.

Using Chem3D (a 3D version of ChemDraw)

a. Deliberately distort the structure of each compound
b. Select Calculations à MM2 à Minimize Energy to conduct the energy
minimization using the most commonly used molecular mechanics force field for
organic compounds called MM2.
c. The Minimize Energy window will open. In the Dynamics tab, adjust the
number of iterations that you would like to run. The default may be on the order
of 10,000, but for simple molecules, a lower number of iterations (e.g. 1,000) may
suffice. Record the parameters that you used in your ELN.
d. Click Run. The run feedback will appear in the bottom panel of the screen and
the structure of the compound will change as the energy minimization takes
place. Your distortion should be corrected by the energy minimization.
e. Save each energy minimized structure in MDL SDfile format.

Using Open Babel

https://open-babel.readthedocs.io/en/latest/Forcefields/mmff94.html

a. In PyRx, use the File → Import (Chemical Table File – SDF) command for each
SDF file (Figure 3). Ligands will be downloaded into PyRx and will appear in the
Open Babel tab on the bottom of the application.
b. Highlight the file that you want to minimize
c. Click on the M icon (Figure 4) to open the energy minimization tool
i. Select the MMFF94 force field
ii. Keep Optimization Algorithm: Conjugate gradients
iii. Total number of steps: set to 1000
iv. Leave all other default values
d. Click OK. The energy minimization should take a few seconds to complete and
the file name will change in the Open Babel window.

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 A B

Figure 4. (A) Icon for energy minimization in Open Babel (PyRx) and (B) selection of the Merck
Molecular Force Field 1994 (MMFF94) force field for energy minimization. Edit the total number of steps
as desired.

11. Upload both ligand files into PyRx, if you have not done so already, by choosing PyRx,
use File → Import (Chemical Table File – SDF) command for the .sdf file (Figure 5).
Ligands will be imported into PyRx and will appear in the Open Babel tab on the bottom
of the application.

Figure 5. Illustration of how to import

SDF files into PyRx

12. In the Open Babel tab, right click one of the ligand files and select Convert All to
Autodock Ligand (.pdbqt) (Figure 6). All the ligands will now be in the Ligands
window on the top of the application under the AutoDock tab.

a. If software stops responding you will need to restart.

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 b. If you are unable to convert all to PDBQT format due to software issues, try to
manually convert several of the ligand files into .pdbqt files. They should appear
in the ligands folder under the AutoDock tab.

Figure 6. How to convert SDF files into PDBQT file format using Open Babel for input into AutoDock
Vina

Docking Experiment

13. Go to the Vina Wizard in the far-left tab of the bottom left window and click the Start
button.

14. In the AutoDock tab of the Navigator window (upper left), click on each ligand and on
the protein file to select them. The Vina Wizard à Select Molecules panel (Figure 7)
will show how many ligands and how many macromolecules you have selected.

Figure 7. Illustration of the Vina Wizard in PyRx

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15. Click the “forward” button in the lower right hand of the Vina window, a white Grid
Box (Figure 8) encasing the protein can be minimized or expanded as needed. Expand
the Grid Box until the whole target is enclosed. You can use the button labelled
“maximized” in the middle of the Vina window to capture the entire molecule.

Figure 8. Grid Box used in AutoDock Vina for

defining the search space of the docking
experiment. The dimensions of the Grid Box
should be recorded in your ELN.

16. Next, click the Run Vina button (on lower left side) to initiate the docking experiment.
The run should take no longer than a few minutes to complete. Once done, the docking
results may be viewed via the Analyze Results tab in the Vina Wizard (Figure 8).

a. Each ligand will be named according to the target’s PDB ID Code followed by a
sequential number based on the order of the listing of the drug in PyRx. For
example, target_2puk_1.cdx is the docking of the first ligand into the protein
with PDB ID Code 2PUK.

b. For a given ligand, there will be several “modes” or conformations computed at

thermodynamic minimum energies. Modes are given arbitrary numbers (0, 1, 2,
3, etc.).

c. Capture the “binding affinity (kcal/mol)” data for each ligand. This data is
actually the DG° value from which you can compute Kd. Data may be saved in
“.csv” format for viewing in Excel using the icon circled in Figure 9.

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 Figure 9. Output from AutoDock Vina as displayed in PyRx

17. Each line item in the Analyze Results window corresponds to a docked ligand file. To
access that file, click the name in the Analyze Results window, which should add it to
the Navigator window in the Molecules tab.

18. In the Molecules tab, right click on the protein name and save it as a PDB file. Then
right click the ligand name and save it as a PDB file also. You will have to save each
conformation of the ligand separately. Click on each different mode in the docking
results list and save the PDB file for each one. You should have 18 PDB ligand files.

Data Analysis
19. Compute Kd values for each mode deduced by AutoDock Vina for each of the two
ligands tested.
a. Tabulate your results with appropriate units and significant figures.
b. Show an example calculation including the equations used and the mathematics
for one example.

20. Visualize the protein ligand complexes in PyMOL

a. Load the protein target in PDB format into PyMOL as a ribbon diagram (i.e.
cartoon rendering)

b. Load the ligand file into the same PyMOL session with the protein target and
render it distinctively (e.g. in space-filling representation)
i. If the ligand file contains all modes in one file, then use the arrows at the
bottom right panel to scroll left or right to view how each mode docks
into the protein target
ii. If each mode for a given ligand is available as a separate file, then
visualize each one by one.

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 c. Identify key domains in the biological target. For a given ligand, describe how
each mode binds to the protein via domains (e.g. N-terminal domain, C-terminal
domain, active site, some other prominent ligand binding site, a functional
domain, etc.)

d. Look for hydrogen bonding, salt bridges and hydrophobic forces that seem to
play a role in stabilizing the ligand in the conformation observed.

e. Generate a figure of the protein-ligand complex showing a representative ligand

at every unique location bound to the protein for drug #1 (Figure A) and drug #2
(Figure B).

21. Deduce any relevant structure-function relationships between the protein and ligands
that support why one drug might be superior to the others of the ATC class. Generate a
figure to illustrate your idea about why one drug is superior to the other given the
structure observed. You should have one figure with A and B images or one figure with the two
different ligands illustrated in the same figure.

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 ADDENDUM
HOW TO ACCESS VLab
• Install Citrix Receiver onto your computer (you can also run a lite version which opens
in your browser).
• Go to mycloud.gatech.edu
• Log in using your GT login and password
• Select DESKTOPS >> COS-2020
• Click Open. Then Citrix Workspace will be launched
• Wait for the desktop to be prepared for you (this takes a couple of minutes)
• The CHEM folder on the desktop should show the following programs:

PyRx (Docking Software)

• Go to https://pyrx.sourceforge.io/
• Select the “Free, 0.8 and earlier” for download
• Note that there is no free version of PyRx for Mac. So Mac users may need to install a
virtual machine for windows system if you are not using VLab.
o Go to http://pwp.gatech.edu/cs2110/wp-
content/uploads/sites/856/2018/12/VM_Installs_Fall_2018_draft2.pdf
o or https://it.iac.gatech.edu/services/vlab for more instructions.

ChemDraw
• ChemBioDraw Ultra 12.0 is preferred. Use GT site license for downloading
• Go to http://www.cambridgesoft.com/software/chembiodraw/
• Note: Mac version lacks Chem3D. Windows version has Chem3D

MGLTools
• Go to http://mgltools.scripps.edu/downloads
• Select the appropriate link for downloading software onto your computer
• Note: Mac users must have X11 software installed. Follow instructions to obtain X11, if
necessary, in order to complete the MGLTools installation.

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 • When the download is complete, you should have 4 programs in your MGL Tools folder
(AutoDock Tools, Python Molecular Viewer [PMV], Update Manager, and Vision)

AutoDock Vina (Docking Software)

• Go to http://vina.scripps.edu/download.html
• Select the appropriate version of the software for download
• Go to http://vina.scripps.edu/tutorial.html and learn the basics of AutoDock Vina
• Explore the other header tabs for more information about AutoDock Vina

PyMOL (Visualization Software)

• Go to https://pymol.org/2/
• Select the appropriate version of the software for download

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