Introduction

The protein c-ABL is mammalian cytplasmic and nuclear protein kinase. In CML(chronic myelogenous leukemia), mutations in the ABL1 gene lead to fusino protein BCR_ABL kinase with unregulated activity. leading to cell prolferation and cancer. Therefore inhibition of the ABL kinase domain is seeen as a viable strategy to mitigate the progression of this disease. The discovery of the first effective BCR_ABL kinase, imatinib, is a classic example of rational design: upon knowledge of the causative mutation, compound libraries were screened for inhbitors of c-ABL knise activity. This discovery spawned a generation of related inhibitors, including dastanib and nilotinib, with more potency and specificity.

In this tutorial, you will learn how to use the molecular docking software Autodock Vina to study the interaction of inhibitor ligands to the c-ABL kinase.

Preparation of files

Open your Virtualbox Linux machine in as previously done (if you don't remember how to do it, ask the instructor).

After the virtual box machine loads, click on the right mouse and in the shown menu choose "Ooen Terminal". Create a folder with the following commands:

You are now ready use PyMOL by executing the command pymol. However, we first need to get the necessary files from the Protein Data Bank and PubChem.

Click on the right mouse button, select "Applications, then "Internet" and then "Firefox". From the Protein Databank Site (www.pdb.org) download the PDB file with code 1iep (imatinib in comlex with human c-ABL kinase). (NOTE: use the option "Save" and not "Open" in Firexo. The file will be loaded to "/home/student/Downloads", and you will have to copy it with the following command on the terminal window:

Open the file in PyMOL with the commandd:

Remove solvent molecules and ions, and also chain B. Identify the ligand (imatinib), select it and save it to pdb file using the command "save imatinib_xtal.pdb, (sele)". Remove the ligand and save the protein (now only chain A) to a file name "kinase.pdb".

Go to PubChem (www.pubchem.org), search for imatinib and download its structure as an sdf file. Open the sdf file in PyMOL,remove its hydrogens ("hydrogens" option in the "A" menu) and save the molecule in a file called "imatinib.pdb". Use "delete all" to delete all molecules in PyMOL, and then load the files "imatinib.pdb" and "kinase.pdb".

Go to the PyMOL top menu "Plugins" and in the entry "Legacy Plugins" choose "Autodock Vina". The Autodock PyMOL plugin window should open, with the following tabs: "Configuration", "Grid Settings", "Recptor", "Ligands", "Docking", "ViewPoses", "Score/Rank" and "Grid Maps". The window should open with the "Configuration" exposed. On that tab, click in "Working Directory" and select the folder you are working from (it shoule be "/home/student/docking").

Now we need to set the parameters for our docking calculation. First, we use the "Grid Settings" menu to define the volume of space to be searched by the docking procedure. We want that volume to encompass the entire protein at first, and preferably to centered on the molecule center of mass. Select the option "Calculate Grid Center by selection" and in the corresponding box type "kinase". In the "Grid Defintion" area use a spacing of 1 and select a box size of 60 (these are distances in Angstroms). Press the button "Show Box" to check the box settings.

Now select the "Receptor", click "receptor kinase" in pymol selections and "Generate receptor".

In the "Ligand" tab repeat the procedure, in this case clicking on the ligand name ("imatinib") from the ligand list and then pressing the button "Generate Ligand".

Preparation of receptor and ligand structures is now complete, and we move to the "Docking" tab to set docking parameters and launch the calculation. In this tab, make sure the receptor box shows "kinase" and the ligand box shows "imatinib". Leave the number of poses at 10 and the "Exaustiveness" parameter to 10. Launch Autodock Vina using the button "Run Vina". The calculation should take a few minutes, you can check its progress on the "Log" box below the run button.

After the run finishes, move the tab "View Poses" to analize the docking results. Click in "Browse" and then select the file "imatinib.docked.pdbqt" which contains the list of 10 docking poses generate by Autodock Vina. In the "Poses" area, we can click on the buttons "Show All" to view or hide all poses, or use the "Docked" box to view individual poses by clicking on them (and see their docked energies). Poses are sorted by docking energy from lowest (most negative) to highest. Lowest energy poses are the best solutions found by the program.

In this case we can check the accuracy of the docking program because we have an actual experimental structure of imatinib docking into c-ABL kinase. That structure was saveb in "imatinib_xtal.pdb". Load this structure and compare it with the found solutions.