Ranking of HIV-protease inhibitors using AutoDock 1. Task Calculate possible binding modes and estimate the binding free energies for 1 3 inhibitors of HIV-protease. You will learn: Some of the theory behind automated docking. To use a docking program that is used by scientists all over the world. To analyze interactions between a ligand and a receptor. 2. A (very) short introduction to AutoDock AutoDock is a program that is designed to dock small molecules to a receptor of known structure. With this program, you can find possible binding sites for ligands and estimate how well they bind to the receptor. In the AutoDock calculations, the receptor is held rigid while the ligand is free to rotate, translate, and change conformation. A modified genetic algorithm (Lamarckian genetic algorithm) is used to identify possible binding modes for the ligand and, during docking, a force field energy function evaluates how well it fits in the receptor. After docking, a semi-empirical energy function is used to estimate the binding free energies for the ligand. 1
3. The receptor: HIV-protease Open a terminal window. (right click on desktop and choose open terminal ) Copy files to your home directory using cp r /ibg/courses/1mb280/proteng/. Open a terminal window and go into to the copied directory (cd ProtEng). Start AutoDock tools by writing adt. Load the receptor in ADT: File Read Molecule. Choose HIVprotease.pdb. Take a close look at the protein using the mouse (See below). Also color the protein using: Color by Atom Type. Try to identify possible binding site! Try to locate a possible binding site for the ligand! Mouse: Right button: Middle button: Middle button+shift: Right button+shift: translation in x,y-direction rotate translation in z-direction changes slab Use Edit Delete Delete Molecule to remove the protein from the screen. Now close ADT. Figure 1. HIV-protease. 2
4. The inhibitors: 1hef, 1heg or 1hvj The pdb files for the ligands are located in the directories: 1hef, 1heg and 1hvj. Move to one of these directories (cd 1xxx)and start adt. Load the ligand using: Ligand Input Molecule Read Molecule. (change file type to *.pdb ) Note that 1hef_lig and 1heg_lig should have an N3+ atom in one end: Edit Atoms Edit Type, click the nitrogen and then choose N3+. Add hydrogens to the chosen ligand by using Edit Hydrogens Add. Save the ligand in its directory as a pdb-file: File Save Write PDB. Name the ligand file:ligand.pdb Use Edit Delete Delete Molecule to remove the molecule from the screen. Make sure that all files have been saved in the correct directory, i.e. the directory of the chosen ligand. Make sure that the ADT window is empty. Ligand Figur 2. 1heg. The module Ligand assigns partial charges, rotatable bonds, aromatic carbons, and identifies polar hydrogens. Reload ligand.pdb using: Ligand Input Molecule Read Molecule - Partial charges are calculated. - Polar hydrogens are identified. - Aromatic carbons are identified. Ligand Define Rigid Root Automatically. Ligand Rotatable Bonds Define Rotatable Bonds Let amide bonds be rigid. All other bonds should be flexible. Look at the screen to identify the bond which will be kept rigid. Make sure that all (and only) amide bonds are Non-rotatable (magenta bonds). Ligand Write PDBQ The above information is written to a file. Name it: ligand.out.pdbq Now remove the ligand from the window! (Edit Delete Delete Molecule) 3
Grid AutoDock uses a grid to calculate the energy of a ligand in the receptor. The grid is calculated before the docking calculation is initialized, which saves computation time. Load HIV-protease using: Grid Macromolecule Read Macromolecule Choose HIVprotease.pdb (The file is located one directory up) - Kollman charges are calculated. - Solvation parameters are assigned. - The molecule is saved as a.pdbqs file. Name it: HIVprotease.pdbqs Choose HIV-protease using: Select Direct Select Molecule list Use File Save Write PDBQS to save the file in the directory of the chosen ligand with the name: HIVprotease.pdbqs. Now press the button: clear selection Identify the atom types of the ligand using Grid Set Map Types By Reading Formatted File. Choose ligand.out.pdbq These atoms will be used as probes in the calculation of AutoDock s grid maps. The window AutoDpf Ligand Parameters shows the identified atom types. Press Accept. Now you will define the volume where the AutoDock will try to dock the ligand. The total number of grid points should be approximately 300 000. Press Grid Set Grid and use the scroll bars to define a suitable position and size of the box. (Do not change the Spacing!) When you are satisfied with the choice of your box, close the window using: File Close saving current. Grid Set Other Options Don t change anything here. Grid Write Gpf Save this file in the directory of the chosen ligand with the name: HIVprotease.gpf Run Run Start AutoGrid With this command you start the program autogrid3, which creates the atom maps that are used in the docking of the inhibitor. Write /opt/bin/autogrid3 in the window Program Pathname and then press enter. Use Launch to start the calculation. Calculating the grid maps takes approximately 60 s and a log file with the name HIVprotease.glg is written. 4
Docking Now we will start the actual docking calculation. Docking Set Macromolecule Choose Macromolecule Choose HIV protease. Docking Set Ligand Parameters Choose Ligand Choose your ligand. The window AutoDpf Ligand Parameters shows your chosen parameters. Click Accept. Docking Set Search Parameters Genetic Algorithm Parameters Everything should be default here except: - Number of GA Runs: 100 - Maximum Number of energy evaluations: 700 000 Docking Set Search Parameters Local Search Parameters: Click Accept. Docking Set Docking Run Parameters: Everything should be set to default except: - RMS Cluster Tolerance (Angstrom): 2.0 Click Accept. Docking Write DPF GALS.dpf Save this file with the name: HIVprotease.dpf Run Run Start AutoDock With this command, the program AutoDock, which performs the actual docking calculation, is started. Write /opt/bin/autodock3 in the window Program Pathname and then press enter. Use Launch to start the calculation. This calculation takes a few hours to complete. You can look in the HIVprotease.dlg file (use tail 100 HIVprotease.dlg) to make sure that the calculation has started. While the calculation is running in the background, set up a new docking for another ligand (close ADT and start from the beginning). The results will be analyzed during your next practical. When you leave, do not log out! Instead, lock your computer by clicking actions and then lock computer on your desktop. 5
5. Analyzing the results Go to the directory containing the ligand. Start ADT by typing adt. Analyze Analyze Docking Logs Read Docking Log Reads the docking results. Analyze Molecules Show Macromolecule Shows the receptor (HIV-protease). Analyze Results In Get Output and Show Histogram the results from the docking are displayed. Analyze Conformations Show Conformations Shows all docked conformations. In Analyze there are also many other ways to analyze the docking results. Explore! For example, the hbondcommands module can be used to show hydrogen bonds. Load it by choosing hbondcommands in File Load Module and press Load Module. To show these hydrogen bonds Build must be used. 6. Some interesting questions to discuss Why and how do these ligands bind to HIV-protease? Which ligand would you suggest as a possible inhibitor? How would you improve it? How does the Lamarckian genetic algorithm work? What assumptions have you made in your calculations? How would you further improve the accuracy of your results? 7. References Morris, G. M., Goodsell, D. S., Halliday, R.S., Huey, R., Hart, W. E., Belew, R. K. and Olson, A. J., Automated Docking Using a Lamarckian Genetic Algorithm and and Empirical Binding Free Energy Function, J. Comp. Chem., 19: 1639-1662, (1998). Murthy, K. H., Winborne, E. L., Minnich, M. D., Culp, J. S., Debouck, C., The crystal structures at 2.2-A resolution of hydroxyethylene-based inhibitors bound to human immunodeficiency virus type 1 protease show that the inhibitors are present in two distinct orientations, J. Biol. Chem., 267, 22770-22778, (1992). Hosur, M. V., Bhat, T. N., Kempf, D. J., Baldwin, E. T., Liu, B. S., Gulnik, S., Wideburg, N. E., Norbeck, D. W., Appelt, K., Erickson, J. W., Influence Of Stereochemistry On Activity and Binding Modes For C(2) Symmetry-Based Diol Inhibitors Of HIV-1 Protease, J. Am. Chem. Soc., 116 847 (1994). 6