Difference between revisions of "2015 DOCK tutorial with Poly(ADP-ribose) polymerase (PARP)"

Revision as of 15:36, 4 March 2015

For additional Rizzo Lab tutorials see DOCK Tutorials. Use this link Wiki Formatting as a reference for editing the wiki. This tutorial was developed collaboratively by the AMS 536 class of 2014, using DOCK v6.6.

I. Introduction

DOCK

DOCK is a molecular docking program used in drug discovery. It was developed by Irwin D. Kuntz, Jr. and colleagues at UCSF (see UCSF DOCK). This program, given a protein binding site and a small molecule, tries to predict the correct binding mode of the small molecule in the binding site, and the associated binding energy. Small molecules with highly favorable binding energies could be new drug leads. This makes DOCK a valuable drug discovery tool. DOCK is typically used to screen massive libraries of millions of compounds against a protein to isolate potential drug leads. These leads are then further studied, and could eventually result in a new, marketable drug. DOCK works well as a screening procedure for generating leads, but is not currently as useful for optimization of those leads.

DOCK 6 uses an incremental construction algorithm called anchor and grow. It is described by a three-step process:

1. Rigid portion of ligand (anchor) is docked by geometric methods.
2. Non-rigid segments added in layers; energy minimized.
3. The resulting configurations are 'pruned' and energy re-minimized, yielding the docked configurations.

1HVR Receptor surface

Poly ADP Ribose Polymerase (PARP)

Poly (ADP-ribose) polymerase (PARP) is a family of proteins involved in a number of cellular processes involving mainly DNA repair and programmed cell death. (Wikipedia: http://en.wikipedia.org/wiki/Poly_ADP_ribose_polymerase) The particular PARP family member we will focus on is PARP5b (aka: Tankyrase 2) of which the catalytic domains contains 227 amino acid residues. Olaparib (AZD-2281, trade name Lynparza) is an FDA-approved chemotherapeutic agent, developed by KuDOS Pharmaceuticals and later by AstraZeneca. It is an inhibitor of poly ADP ribose polymerase (PARP), an enzyme involved in DNA repair.[1] It acts against cancers in people with hereditary BRCA1 or BRCA2 mutations, which includes many ovarian, breast, and prostate cancers. (Wikipedia: http://en.wikipedia.org/wiki/Olaparib)

In this class, we will perform docking experiments and virtual screening on a crystallographic structure of PARP5b in complex with a small-molecule inhibitor, olaparib (PDB ID: 4TKG).

Organizing Directories

While performing docking, it is convenient to adopt a standard directory structure / naming scheme, so that files are easy to find / identify. For this tutorial, we will use something similar to the following:

~username/AMS536/dock-tutorial/00.files/
                              /01.dockprep/
                              /02.surface-spheres/
                              /03.box-grid/
                              /04.dock/
                              /05.mini-virtual-screen/
                              /06.virtual-screen/
                             

In addition, most of the important files that are derived from the original crystal structure will be given a prefix that is the same as the PDB code, '4TKG'. The following sections in this tutorial will adhere to this directory structure/naming scheme.

II. Preparing the Receptor and Ligand

Go to the Protein Databank Website (pdb.org) and search for 4TKG. This is the code for PARP protein crystal structure in complex with olaparib. Download the PDB (text) file for this protein. You will then want go to your /00.files directory and copy this file using the command below.

 cp ~/Downloads/4TKG.pdb ./

And then we will create 4 files in 01.dockprep/ directory:

 4TKG.dockprep.mol2  
 4TKG.ligand.mol2  
 4TKG.receptor.mol2  
 4TKG.receptor.noH.pdb

Create the dockprep file

To create the 4TKG.dockprep.mol2 file, you will first need to open 4TKG.pdb in Chimera. You will notice that there are four copies of this protein-ligand complex in the original crystal structure. Since we only want to work with one of these, select 'Chain A' (select->chain->A). Once Chain A is selected we will invert the selection by going the the 'Select' tab-> invert(all models). Next we will delete these chains by going to the 'Actions' tab->atoms/bonds->delete. Save this file as 4TKG.dockprep.mol2 in your 01.dockprep directory.

For the "1HVR.dockprep.mol2" file: open the 1HVR.modified.pdb in Chimera; delete the water molecules ; delete the original hydrogen atoms; add the charge by go to Tools->structure editing->add charge (Note when adding the charge to the ligand, you can choose AMBER ff99SB as the charge model and chose gasteiger as the charge method. In this 1HVR case, we set Net Charge to 0. You may have to consider the chemistry of the ligand when assigning a charge state). Add the hydrogen atoms manually by Tools->structure editing->Add H . Or you can do all of the above by clicking Tools -> Structure Editing -> Dock Prep.

III. Generating Receptor Surface and Spheres

Generating the Receptor Surface

Xingyu

Placing Spheres

Sphgen is a program that generate sets of overlapping spheres that define the shape of a molecule or molecular surface. Spheres are generated over the entire receptor and ligand surface. For further information on how Sphgen functions, please refer to the latest version of the DOCK manual:

<http://dock.compbio.ucsf.edu/DOCK_6/dock6_manual.htm>

To generate spheres using Sphgen follow the steps below:

Step 1. Create an input file name INSPH with the following information:

vim INSPH

4TKG.rec.dms #surface file generated above will be the input file
R            #flag to place spheres outside (R) or inside (L) of the surface 
X            #flag that informs sphgen of the subset of surface points to be used (X = all points)
0.0          #flag that prevent the generation of large spheres with close surface contacts(default= 0)
4.0          #maximum radius of the spheres generated (default = 4.0 Angstroms)
1.4          #minimum radius of the spheres generated (default = radius of probe)
4TKG.rec.sph #this will be the file which contained the clustered spheres generated

Step 2. Run the program Sphgen using the command sphgen:

sphgen -i INSPH -o OUTSPH

-i is the flag that give sphgen the input file INSPH
INSPH is the file created above that gives sphgen instructions
-o is the flag to create the oputput file
OUTSPH is the output file with the information of the spheres generated from sphgen

Step 3. Visualization of the spheres generated:

Visualization of the spheres can be done directly with chimera or with the program showsphere

3 a. Visualization directly with Chimera:

• Launch Chimera, choose File -> Open, choose 4TKG.rec.mol2
• choose File -> Open, choose 4TKG.rec.sph

You should have an image like this:

4TKG Receptor surface (light gray) with all spheres (various colors) generated

3 b. Visualization with showsphere:

showsphere convert the .sph file into PDB format.

(i) Run showsphere, by typing showsphere into the terminal:

showsphere

You will be prompted with the following questions:

Enter name of sphere cluster file:
     4TKG.rec.sph
Enter cluster number to process (<0 = all):
     -1
Generate surfaces as well as pdb files (<N>/Y)?
     N
Enter name for output file prefix:
     output_spheres
Process cluster 0 (contains ALL spheres) (<N>/Y)? 
     N

-1 is a flag that allow you to see all possible spheres

(ii) Open Chimera

• Launch Chimera, choose File -> Open, choose 4TKG.rec.noH.pdb
• Go File -> Open, choose output_spheres.pdb

You should see many spheres placed all over the receptor surface.

4TKG Receptor surface (light gray) with all spheres (various colors) using the program showsphere for visualization. The Yellow spheres are located in the binding site of the receptor

Step 4. Selecting spheres of interest:

To select spheres of interest you need to run a program name sphere_selector in the terminal. The idea is to allow the program to select spheres that are within a user-defined radius (in this case, 8.0 angstroms) of a target molecule or a known binding site:

sphere_selector 4TKG.rec.sph ../01.dockprep/4TKG.lig.mol2 8.0

A new file name selected_spheres.sph will be generated.

Step 5. Visualize the spheres using showsphere as previously done:

showsphere

When prompted on the command line, answering the questions as follows:

Enter name of sphere cluster file:
     selected_spheres.sph
Enter cluster number to process (<0 = all):
     -1
Generate surfaces as well as pdb files (<N>/Y)?
     N
Enter name for output file prefix:
     output_spheres_selected
Process cluster 0 (contains ALL spheres) (<N>/Y)? 
     N

View spheres in Chimera:

• Launch Chimera, choose File -> Open, choose 4TKG.rec.noH.pdb
• Go File -> Open, choosing output_spheres_selected.pdb
• Go Select -> Residue -> SPH
• Go, Actions -> Atoms/Bonds -> sphere

4TKG Receptor surface (light gray) with spheres (blue) within 8A

IV. Generating Box and Grid

Box Generation

• Make a new directory and name it: 03.box-grid/

   mkdir 03.box-grid

• Make a new file in this directory and name it showbox.in

   vim showbox.in

• This will automatically open the file showbox.in. Edit the file showbox.in as follows:

   Y                                               # Yes, generate a box
   8.0                                             # Size of the box in Angstroms
   ../02.surface-spheres/selected_spheres.sph      # Sphere.sph file
   1                                               # Cluster number
   4TKG.box.pdb                                    # Name of the output file

• Save the file using the command:

    :wq

• Run the command:

    showbox < showbox.in

Selected sphere surface with generated box

Cong Liu

Grid computing

In order to save computational resources and speed up the docking process, we let dock to pre-calculate the potential energy around the docking region which defined by previous section before we perform docking calculation. In grid program, there are two ways to evaluate the potential energy in docking region: contact and energy scoring. The users could apply these two method independently to their docking system by simply typing “yes/no” in the input file grid.in. Once finished, the grid results will be saved in the corresponding extension file: *.cnt and *.nrg. Another important parameter in grid program is bump grid. This variable determines the degree of overlapping among receptor's atoms. The usage method is same as contact or energy scoring.

In this tutorial, we just use the energy scoring option to evaluate the potential energy in docking region. Mathematically grid use the empirical London-Jone's model and Coulomb electrostatic interaction function to approximate the potential energy in each grid points. The coefficient for electrostatic interaction is fixed. However, you could specify the exponent order for vdw interaction calculation(a and b) by setting the attractive_exponent and repulsive_exponent variable value. Other coefficients in London-Jone model are specified by the vdw_AMBER_parm99.defn file.

In practice, you have two ways to calculate the grid. The more clear and efficient way:

• Create the grid input file:

 vi grid.in

• Setting all variables' value in grid.in. And run the program:

 grid -i grid.in

A more interactive and user friendly way:

• Run the grid program

 grid

Then follow the instruction of the program and setting all variables by answering each questions. If it were you first time to run dock, we strongly recommend you to use second method.

For this tutorial, the Table summarize all parameters that will be needed and give a brief description.

Table grid.in

More detail, please click :

http://dock.compbio.ucsf.edu/DOCK_6/tutorials/grid_generation/generating_grid.html

http://dock.compbio.ucsf.edu/DOCK_6/dock6_manual.htm#GridOverview

V. Docking a Single Molecule for Pose Reproduction

Michael Cortes

Beibei Zhang

Prajna Shanbhogue

Now, run:

dock6 -i rgd.in

You will now be required to answer a series of questions regarding location of input file, docking parameters and output file prefix. Answer all the questions as follows:

ligand_atom_file                                             ../01.dockprep/4TKG.lig.mol2

limit_max_ligands no skip_molecule no read_mol_solvation no calculate_rmsd yes use_rmsd_reference_mol no use_database_filter no orient_ligand yes automated_matching yes receptor_site_file ../02.surface-spheres/selected_spheres.sph max_orientations 1000 critical_points no chemical_matching no use_ligand_spheres no use_internal_energy yes internal_energy_rep_exp 12 flexible_ligand no bump_filter no score_molecules yes contact_score_primary no contact_score_secondary no grid_score_primary yes grid_score_secondary no grid_score_rep_rad_scale 1 grid_score_vdw_scale 1 grid_score_es_scale 1 grid_score_grid_prefix ../03.box-grid/grid multigrid_score_secondary no dock3.5_score_secondary no continuous_score_secondary no descriptor_score_secondary no gbsa_zou_score_secondary no gbsa_hawkins_score_secondary no SASA_descriptor_score_secondary no amber_score_secondary no minimize_ligand yes simplex_max_iterations 1000 simplex_tors_premin_iterations 0 simplex_max_cycles 1 simplex_score_converge 0.1 simplex_cycle_converge 1.0 simplex_trans_step 1.0 simplex_rot_step 0.1 simplex_tors_step 10.0 simplex_random_seed 0 simplex_restraint_min no atom_model all vdw_defn_file ../zzz.parameters/vdw_AMBER_parm99.defn flex_defn_file ../zzz.parameters/flex.defn flex_drive_file ../zzz.parameters/flex_drive.tbl ligand_outfile_prefix 4TKG.rgd write_orientations no num_scored_conformers 5000 write_conformations no cluster_conformations yes cluster_rmsd_threshold 2.0 rank_ligands no

VI. Virtual Screening

george jones

Sam Chiappone

VIII. Frequently Encountered Problems