Introductory Tutorial for the UCSF DOCK Program (v. 6.1)

Transcription

1 Introductory Tutorial for the UCS DCK Program (v. 6.1) Author: John E. Kerrigan, Ph.D. University of Medicine & Dentistry of ew Jersey Robert Wood Johnson Medical School 675 oes Lane Piscataway, J U.S.A. (732) phone (732) fax kerrigje@umdnj.edu 1

2 rganization of Workspace: Create a project directory (use the unix mkdir command). Use this directory as your working directory. mkdir proj1 cd proj1 CAUTI: Be sure that you have eugh disk quota for your docking job. The dms program, sphgen and DCK use a fair amount of disk space. To check your quota use the following commands: If logged on to one of the linux workstations 1 st login to the main server to check your quota. ssh username@server.umdnj.edu quota v Exit when finished. Type exit then hit the Enter key. Docking in DCK is divided into four stages: STAGE 1. Ligand Preparation STAGE 2. Site Characterization STAGE 3. Scoring Grid Calculation STAGE 4. Docking Download the dock_input.tgz file from the course webpage ( ow DCK works in a nutshell. irst, you begin with an x-ray crystal structure of a drug/receptor complex. The active site is identified or defined a priori. Points within this site kwn as spheres are used to define the volume or space within the active site pocket where the drug binds. The purpose of the spheres is to generate an unbiased grid of sphere centers that reflects the actual shape of the active site (i.e. the protein/macromolecule dictates the shape of the pocket; t the drug) using the grid program. [1, 2] Sphere centers are matched with ligand (drug) atoms to generate orientations of the ligand in the active site within the program DCK. [2, 3] The orientation of the ligand is scored using a shape-scoring function and/or an energy function (e.g. E bind = E vdw + E elec ). The shape score is an empirical van der Waals attractive energy. As a final step, the orientation may be energy minimized using a rigid-body simplex minimization. [4] Research Problem: Inhibition of actor Xa We will study the inhibition of coagulation factor, factor Xa, as this enzyme has a multitude of inhibitor data. The x-ray crystal structure (1JS) will be used as a modeling template. [5] STAGE 1. Prepare the ligand. 2

3 btain the PDB file, 1JS.pdb, from the protein data bank ( ). We will recommend the UCS Chimera software package [6] for build of the ligand and protein (receptor) models from the x-ray crystal structure coordinates (PDB file). owever, we need to set special ACR and RIGID atom sets. or this we need to use the Sybyl (Tripos, Inc.) software package. pen Sybyl and perform the following ile > Read Read ile > iles: (select 1JS.pdb) > ile Type: M1: (select with mouse) > ile to read: 1JS.pdb (select with mouse) > Click K > ption Center the Molecule > Click CETER_VIEW > K Use the middle mouse button to translate the structure and the right mouse button to rotate the structure. Build/Edit > Extract Atom Expression > Substructures > thers: A/Z34500 > Click K and K Molecule Area > M2: > Click K Remove the enzyme from display area M1: Build/Edit > Zap (Delete) Molecule > Molecule Expression > Select M1: click K Zoom into the drug using the middle and right mouse buttons (press simultaneously) and use the middle mouse button to translate (center). Color by atom type. View > Color > By Atom Type ame the drug molecule Build/Edit > ame Molecule > ZK > K Set the Depth Cue to zero using the depth cue button on the left hand menu. Label atoms by type using the checkmark button on the left hand menu (your drug is in display area D2) There are several atom types we must fix before proceeding. 3

4 Initial Atom Types Build/Edit > Modify > Atom > ption > LY_TYPE Click K Atom Expression > Using the Mouse: Select all atoms requiring atom type correction (see the final corrected atom types in the figure below). Your atom type labels should appear as in the figure below. ow add the hydrogen atoms. Corrected Atom Types 4

5 Build/Edit > Add > ydrogens Compute > Charges > Gasteiger-uckel Click on o when the dialog asks you if you want to change formal charges. Before you save the ligand, you must declare the Anchor atoms as a static set and declare the certain bonds in the structure as RIGID. The ACR set Build/Edit > Define > Static Set > ption > ATM Click K In Atom Expression, select the following asterisked atoms * * ame this set ACR. * C 3 C 3 The RIGID set Build/Edit > Define > Static Set > ption > BD Click K In Bond Expression, select the following highlighted bond C 3 C 3 ame this set RIGID. Using ile > Save As Save the ligand as zk mol2; ormat: ML2. 5

6 STAGE 2. Site Characterization. We must remove the ligand and any solvent from the receptor (protein). We will use chimera for preparation of the receptor. Start Chimera by typing chimera in the command line in the unix shell. Load 1JS.pdb. ile > pen > 1JS.pdb Delete the nstandard residues (water, ligand, etc.). Select > Residue > all nstandard Actions > Atoms/Bonds > delete Use the Dock Prep tool in Structure editing to prepare the mol2 file. Tools > Structure Editing > Dock Prep Add hydrogens 6

7 Save the mol2 file Edit the insertion code residue ID numbers. Insertion code is used to keep residue numbering consistent from species to species. owever, it can be a pain for molecular modeling programs. The sphgen program will choke on insertion code residue ID numbers as these numbers have a letter appended to them (e.g. 61A). Edit the fxa.pdb file in any text editor and remove the letters from these residue ID numbers (replace with a space ). Run the dms program and create the molecular surface data for the sphere calculation. Type man dms to obtain more information about the dms program and its run options. dms fxa.pdb a n w 1.4 v o fxa.ms ext, we must run sphgen. In order to do so, we must have a file kwn as ISP as input for the sphgen program. The contents of ISP are as follows (nly use the information between the hashed lines!). ISP fxa.ms R X fxa.sph Run sphgen simply by typing sphgen in the unix shell as follows. sphgen 7

8 ext we will use the coordinates of the ligand from the crystal structure to select the relevant spheres for the grid and docking computations. or this we will use a program called sphere_selector. The command line format is: sphere_selector file.sph ligand.mol2 #.# where #.# is the number of angstroms out from the ligand that you want to include spheres. sphere_selector fxa.sph zk mol2 6.0 The output from this operation is always a file named selected_spheres.sph. Use the showsphere program to make a pdb file of the selected spheres. Type showsphere <enter> ame of the sphere cluster file: selected_spheres.sph Cluster number to process: 1 Generate surfaces? ame for output pbd file: sel_sph.pdb Spheres selected by the sphere_selector program in red. Run showbox. 8

9 Type showbox <enter>. automatically construct box to enclose spheres [Y/]? Y extra margin to also be enclosed (angstroms)? 10 sphere fileselected_spheres.sph cluster number 1 output filename? site_box.pdb STAGE 3. Scoring Grid Calculation Download grid.in file from the course web page and use it for your grid input. See contents of grid.in which follows. Run grid. hup grid i grid.in o grid.out > grid.out & Contents of grid.in compute_grids grid_spacing 0.3 output_molecule contact_score energy_score energy_cutoff_distance 9999 atom_model a attractive_exponent 6 repulsive_exponent 12 distance_dielectric dielectric_factor 4 bump_filter bump_overlap 0.75 receptor_file fxa.mol2 box_file site_box.pdb vdw_definition_file /usr/local/dock6/parameters/vdw_amber_parm99.defn score_grid_prefix grid STAGE 4. Docking ow run DCK. Download dock.in file from the course web page and use it for you dock input. See the dock.in file which follows. 9

10 Run DCK using the following command hup dock6 i dock.in o dock.out > dock.out & Contents of dock.in ligand_atom_file zk mol2 limit_max_ligands skip_molecule read_mol_solvation calculate_rmsd orient_ligand automated_matching receptor_site_file selected_spheres.sph max_orientations 500 critical_points chemical_matching use_ligand_spheres flexible_ligand min_anchor_size 40 pruning_use_clustering pruning_max_orients 100 pruning_clustering_cutoff 100 use_internal_energy internal_energy_att_exp 6 internal_energy_rep_exp 12 internal_energy_dielectric 4.0 use_clash_overlap bump_filter score_molecules contact_score_primary contact_score_secondary grid_score_primary grid_score_secondary grid_score_rep_rad_scale 1 grid_score_vdw_scale 1 grid_score_es_scale 1 grid_score_grid_prefix grid minimize_ligand minimize_anchor minimize_flexible_growth use_advanced_simplex_parameters 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_anchor_max_iterations 500 simplex_grow_max_iterations 500 simplex_final_min simplex_secondary_minimize_pose simplex_random_seed 0 atom_model all vdw_defn_file /usr/local/dock6/parameters/vdw_amber_parm99.defn flex_defn_file /usr/local/dock6/parameters/flex.defn flex_drive_file /usr/local/dock6/parameters/flex_drive.tbl ligand_outfile_prefix zk807_out write_orientations num_primary_scored_conformers_rescored 1 num_secondary_scored_conformers_written 1 rank_primary_ligands rank_secondary_ligands 10

11 ere are our results rom the tail of dock.out (tail -25 dock.out): Molecule: ZK Secondary Score Grid Score: vdw: es: Molecules Processed Total elapsed time: 5764 seconds The structure in gray is from the x-ray crystal structure and the structure in green is the docked structure. rom the figure above we see that by and large DCK has returned the same orientation as from the crystal structure. There are a few differences in conformation, however. ext, we will dock a small database of compounds (we used the zk compound from the crystal structure as a template to build these compounds in Sybyl 7.0; hence, zk also appears in this database). The database is in mol2 format. Download pyridins.mol2 from the course webpage. 11

12 Contents of dock_db.in ligand_atom_file pyridins.mol2 limit_max_ligands skip_molecule read_mol_solvation calculate_rmsd orient_ligand automated_matching receptor_site_file selected_spheres.sph max_orientations 500 critical_points chemical_matching use_ligand_spheres flexible_ligand min_anchor_size 40 pruning_use_clustering pruning_max_orients 100 pruning_clustering_cutoff 100 use_internal_energy internal_energy_att_exp 6 internal_energy_rep_exp 12 internal_energy_dielectric 4.0 use_clash_overlap bump_filter score_molecules contact_score_primary contact_score_secondary grid_score_primary grid_score_secondary grid_score_rep_rad_scale 1 grid_score_vdw_scale 1 grid_score_es_scale 1 grid_score_grid_prefix grid minimize_ligand minimize_anchor minimize_flexible_growth use_advanced_simplex_parameters 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_anchor_max_iterations 500 simplex_grow_max_iterations 500 simplex_final_min simplex_secondary_minimize_pose simplex_random_seed 0 atom_model all vdw_defn_file /usr/local/dock6/parameters/vdw_amber_parm99.defn flex_defn_file /usr/local/dock6/parameters/flex.defn 12

13 flex_drive_file /usr/local/dock6/parameters/flex_drive.tbl ligand_outfile_prefix pyridins_out write_orientations num_primary_scored_conformers_rescored 1 num_secondary_scored_conformers_written 1 rank_primary_ligands max_primary_ranked 500 rank_secondary_ligands hup dock6 i dock_db.in o dock.out > dock_db.out & The database docking gives slightly different results for the zk compound. This run was performed on a 2.2 Gz Linux workstation. ere are the structures of the compounds in the database: * * * C 3 C 3 2 CMPD 001 C 2 C 3 CMPD 002 C 3 C 3 ZK C 3 CMPD V CMPD IV 13

14 Ranked Scores: Molecule: CMPD_V Secondary Score Grid Score: vdw: es: Molecule: ZK Secondary Score Grid Score: vdw: es: Molecule: CMPD_IV Secondary Score Grid Score: vdw: es: Molecule: CMPD002 Secondary Score Grid Score: vdw: es: Molecule: CMPD001 Secondary Score Grid Score: vdw: es: Molecules Processed Total elapsed time: seconds The pyridins_out_scored.mol2 database can be viewed using chimera (see or Sybyl. CMPD CMPD CMPD_IV CMPD_V ZK

15 y = x R 2 = Eint (kcal/mol) log Ki The K i data for compounds 001 and 002 were obtained from a review paper. [7] All other K i data were obtained from the structure paper. [5] ur principal outlier is compound ZK The other compounds in the series fall into line in this plot very well. ormally, we would sample more than just five compounds to establish a trend. This example is just illustrative of the application of the DCK method and to caution one about the use of the simple scoring function (E int = E vdw + E elec ) used in DCK. References: 1. Meng, E.C., B.K. Shoichet, and I.D. Kuntz, Automated docking with grid-based energy evaluation. J. Comp. Chem., : p Shoichet, B.K., D.L. Bodian, and I.D. Kuntz, Molecular docking using shape descriptors. J. Comp. Chem., (3): p Kuntz, I.D., et al., A geometric approach to macromolecule-ligand interactions. J. Mol. Biol., : p Meng, E.C., et al., rientational sampling and rigid-body minimization in molecular docking. Proteins, (3): p Adler, M., et al., Preparation, Characterization and the Crystal Structure of the Inhibitor Zk (Ci-1031) Complexed with actor Xa. Biochemistry, (41): p Pettersen, E., et al., UCS Chimera - A Visualization System for Exploratory Research and Analysis. J. Comput. Chem., : p Kontogiorgis, C. and D. adjipavlou-litina, Current Trends in Quantitative Structure Activity Relationships on Xa inhibitors: Evaluation and Comparative Analysis. Med. Res. Rev., (6): p