View source for 2024 Denovo tutorial 1 with PDBID 2ITO


=Introduction=
DeNovo Design aims to create a new ligand from scratch. It is incrementally designed bad added known structures of bioacitve compounds. The end result will hopefully produce a new ligand that can bind more tightly to a protein receptor than the current ligand can. There are three methods that will be introduced in this tutorial:
#Generic DeNovo Design
#Focused Fragment Design
#DeNovo Refinement

We will do denovo design with PDB: 2ITO.


==Setting Up Your Environment==
We will add more directories to the ones we used from the traditional virtual screening.

  009_deNovoRefinement
  010a_fragLib   
  010b_focusGrowth
  010c_focusReScore
  011_genericDeNovo

=DeNovo Refinement=
DeNovo Refinement can be used to assess the effects upon ligand and protein interactions by chaning a portion of the molecule. We can experiment with an interesting piece of the ligand and delete the remaining portion. This deleted portion needs to be replaced with a dummy atom. Dock will then find which fragments can be placed in this position that will have the least energy. 

==Setting up the dummy atom==


==Running DeNovo Refinement==


==Viewing New Molecules==

=Focused DeNovo Design=

==Fragment Library Generation==

==DeNovo Design==

In this next step we will be navigating to the working directory for deNovo design using the Focus algorithm. The name of the directory for working would be 013b.foucusGrowth. As a sequel step from identifying the fragment library in the previous step, it would be required that you already achieved the library fragments set and store it correctly in the 013a.fragLib.

Initiating the input file with vi: 

 vi deNovoFocus.in

Insert the following content for the input:

 conformer_search_type                                        denovo
 dn_fraglib_scaffold_file                                     ../013a.fragLib/2tio_fragLib_scaffold.mol2
 dn_fraglib_linker_file                                       ../013a.fragLib/2tio_fragLib_linker.mol2
 dn_fraglib_sidechain_file                                    ../013a.fragLib/2tio_fragLib_sidechain.mol2
 dn_user_specified_anchor                                     no
 dn_use_torenv_table                                          yes
 dn_torenv_table                                              ../013a.fragLib/2tio_fragLib_torenv.dat
 dn_sampling_method                                           graph
 dn_graph_max_picks                                           30
 dn_graph_breadth                                             3
 dn_graph_depth                                               2
 dn_graph_temperature                                         100.0
 dn_pruning_conformer_score_cutoff                            100.0
 dn_pruning_conformer_score_scaling_factor                    1.0
 dn_pruning_clustering_cutoff                                 100.0
 dn_constraint_mol_wt                                         550.0
 dn_constraint_rot_bon                                        15
 dn_constraint_formal_charge                                  2.0
 dn_heur_unmatched_num                                        1
 dn_heur_matched_rmsd                                         2.0
 dn_unique_anchors                                            2
 dn_max_grow_layers                                           9
 dn_max_root_size                                             25
 dn_max_layer_size                                            25
 dn_max_current_aps                                           5
 dn_max_scaffolds_per_layer                                   1
 dn_write_checkpoints                                         yes
 dn_write_prune_dump                                          no
 dn_write_orients                                             no
 dn_write_growth_trees                                        yes
 dn_output_prefix                                             2tio_focused
 use_internal_energy                                          yes
 internal_energy_rep_exp                                      12
 internal_energy_cutoff                                       100.0
 use_database_filter                                          no
 orient_ligand                                                yes
 automated_matching                                           yes
 receptor_site_file                                           ../002.surface/selected_spheres.sph
 max_orientations                                             1000
 critical_points                                              no
 chemical_matching                                            no
 use_ligand_spheres                                           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                                       ../003.gridbox/grid
 multigrid_score_secondary                                    no
 dock3.5_score_secondary                                      no
 continuous_score_secondary                                   no
 footprint_similarity_score_secondary                         no
 pharmacophore_score_secondary                                no
 descriptor_score_secondary                                   no
 gbsa_zou_score_secondary                                     no
 gbsa_hawkins_score_secondary                                 no
 SASA_score_secondary                                         no
 amber_score_secondary                                        no
 minimize_ligand                                              yes
 minimize_anchor                                              yes
 minimize_flexible_growth                                     yes
 use_advanced_simplex_parameters                              no
 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_grow_tors_premin_iterations                          0
 simplex_random_seed                                          0
 simplex_restraint_min                                        no
 atom_model                                                   all
 vdw_defn_file                                                /gpfs/projects/AMS536/zzz.programs/dock6.10/parameters/vdw_de_novo.defn
 flex_defn_file                                               /gpfs/projects/AMS536/zzz.programs/dock6.10/parameters/flex.defn
 flex_drive_file                                              /gpfs/projects/AMS536/zzz.programs/dock6.10/parameters/flex_drive.tbl

Start running the deNovo focus algorithm with the input as: 

 dock6 -i deNovoFocus.in -o deNovoFocus.out

After running the command, you should leave your program run until it executes. The program could not be run on server with mpi run. After you have successfully run the command, please check the directory output file to see if there have been any errors and the directory should contain multiple mol2 files in which the build.mol2 would be summarizing the molecules that have been built with denovo design algorithm.

==ReScoring Designed Molecules==

In other to make the multiples molecules suggested in the last step become more informative on which molecule would be the most likely to become the drug lead, in this step, the molecule set would be re-scored so that each would be compared with the binding of the original binding ligand to see if they would govern a similar effect to the original binder. The algorithm used for this scoring would be footprint similarity score, to begin, we would navigate to the directory designated for this section 013c.focusReScore. Here please initiate by creating the input file:

 vi focusReScore.in

Insert the following content for the input:

conformer_search_type                                        rigid
 use_internal_energy                                          yes
 internal_energy_rep_exp                                      12
 internal_energy_cutoff                                       100.0
 ligand_atom_file                                             ../013b.focusGrowth/2tio_focused.denovo_build.mol2
 limit_max_ligands                                            no
 skip_molecule                                                no
 read_mol_solvation                                           no
 calculate_rmsd                                               no
 use_database_filter                                          no
 orient_ligand                                                no
 bump_filter                                                  no
 score_molecules                                              yes
 contact_score_primary                                        no
 contact_score_secondary                                      no
 grid_score_primary                                           no
 grid_score_secondary                                         no
 multigrid_score_primary                                      no
 multigrid_score_secondary                                    no
 dock3.5_score_primary                                        no
 dock3.5_score_secondary                                      no
 continuous_score_primary                                     no
 continuous_score_secondary                                   no
 footprint_similarity_score_primary                           no
 footprint_similarity_score_secondary                         no
 pharmacophore_score_primary                                  no
 pharmacophore_score_secondary                                no
 descriptor_score_primary                                     yes
 descriptor_score_secondary                                   no
 descriptor_use_grid_score                                    no
 descriptor_use_multigrid_score                               no
 descriptor_use_continuous_score                              no
 descriptor_use_footprint_similarity                          yes
 descriptor_use_pharmacophore_score                           yes
 descriptor_use_tanimoto                                      yes
 descriptor_use_hungarian                                     yes
 descriptor_use_volume_overlap                                yes
 descriptor_fps_score_use_footprint_reference_mol2            yes
 descriptor_fps_score_footprint_reference_mol2_filename       ../004.energy_min/2tio.lig.min_scored.mol2
 descriptor_fps_score_foot_compare_type                       Euclidean
 descriptor_fps_score_normalize_foot                          no
 descriptor_fps_score_foot_comp_all_residue                   yes
 descriptor_fps_score_receptor_filename                       ../001.structure/2tio_protein_hydrogens_charges.mol2
descriptor_fps_score_vdw_att_exp                             6
 descriptor_fps_score_vdw_rep_exp                             12
 descriptor_fps_score_vdw_rep_rad_scale                       1
 descriptor_fps_score_use_distance_dependent_dielectric       yes
 descriptor_fps_score_dielectric                              4.0
 descriptor_fps_score_vdw_fp_scale                            1
 descriptor_fps_score_es_fp_scale                             1
 descriptor_fps_score_hb_fp_scale                             0
 descriptor_fms_score_use_ref_mol2                            yes
 descriptor_fms_score_ref_mol2_filename                       ../004.energy_min/2tio.lig.min_scored.mol2
 descriptor_fms_score_write_reference_pharmacophore_mol2      no
 descriptor_fms_score_write_reference_pharmacophore_txt       no
 descriptor_fms_score_write_candidate_pharmacophore           no
 descriptor_fms_score_write_matched_pharmacophore             no
 descriptor_fms_score_compare_type                            overlap
 descriptor_fms_score_full_match                              yes
 descriptor_fms_score_match_rate_weight                       5.0
 descriptor_fms_score_match_dist_cutoff                       1.0
 descriptor_fms_score_match_proj_cutoff                       0.7071
 descriptor_fms_score_max_score                               20
 descriptor_fingerprint_ref_filename                          ../004.energy_min/2tio.lig.min_scored.mol2
 descriptor_hms_score_ref_filename                            ../004.energy_min/2tio.lig.min_scored.mol2
 descriptor_hms_score_matching_coeff                          -5
 descriptor_hms_score_rmsd_coeff                              1
 descriptor_volume_score_reference_mol2_filename              ../004.energy_min/2tio.lig.min_scored.mol2
 descriptor_volume_score_overlap_compute_method               analytical
 descriptor_weight_fps_score                                  1
 descriptor_weight_pharmacophore_score                        1
 descriptor_weight_fingerprint_tanimoto                       -1
 descriptor_weight_hms_score                                  1
 descriptor_weight_volume_overlap_score                       -1
 gbsa_zou_score_secondary                                     no
 gbsa_hawkins_score_secondary                                 no
 SASA_score_secondary                                         no
 amber_score_secondary                                        no
 minimize_ligand                                              no
 atom_model                                                   all
 vdw_defn_file                                                /gpfs/projects/AMS536/zzz.programs/dock6.10/parameters/vdw_de_novo.defn
 flex_defn_file                                               /gpfs/projects/AMS536/zzz.programs/dock6.10/parameters/flex.defn
 flex_drive_file                                              /gpfs/projects/AMS536/zzz.programs/dock6.10/parameters/flex_drive.tbl
 chem_defn_file                                               /gpfs/projects/AMS536/zzz.programs/dock6.10/parameters/chem.defn
 pharmacophore_defn_file                                      /gpfs/projects/AMS536/zzz.programs/dock6.10/parameters/ph4.defn
ligand_outfile_prefix                                        2tio_focus_rescore
 write_footprints                                             yes
 write_hbonds                                                 yes
 write_orientations                                           no
 num_scored_conformers                                        1
 rank_ligands                                                 no

Start running the rescore algorithm with the input as: 

 dock6 -i focusReScore.in -o focusReScore.out

If you have successfully computed the simulation you should receive the output results as followed:
	4s0v_focus_rescore_footprint_scored.txt
	4s0v_focus_rescore_hbond_scored.txt
	4s0v_focus_rescore_scored.mol2

These are the sets of molecules suggested by deNovo being ranked based on the footprint scores (as mentioned above), and specifically with the hydrogen bonding scores. In other to view the results, you would need to load the result mol2 attached down to your local computer and view that with Chimera.

Below is loading the mol2 with ViewDock and ranking the file based on the footprint similarity score:

[[File:Focus1png.png]]

[[File:Focus2png.png]]

=Generic DeNovo Design=

For generic denovo design, we are not limiting DOCK to build new molecules using fragments present in the original ligand. Instead, we will be taking fragments from a fragment library. 

Create the input file: 

  vi 2ito_generic.in

The file should contain these lines:

  conformer_search_type                                        denovo
  dn_fraglib_scaffold_file                                     /gpfs/projects/AMS536/zzz.programs/dock6.10/parameters/fraglib_scaffold.mol2
  dn_fraglib_linker_file                                       /gpfs/projects/AMS536/zzz.programs/dock6.10/parameters/fraglib_linker.mol2
  dn_fraglib_sidechain_file                                    /gpfs/projects/AMS536/zzz.programs/dock6.10/parameters/fraglib_sidechain.mol2
  dn_user_specified_anchor                                     no
  dn_torenv_table                                              /gpfs/projects/AMS536/zzz.programs/dock6.10/parameters/fraglib_torenv.dat
  dn_name_identifier                                           2ito_generic
  dn_sampling_method                                           graph
  dn_graph_max_picks                                           30
  dn_graph_breadth                                             3
  dn_graph_depth                                               2
  dn_graph_temperature                                         100.0
  dn_pruning_conformer_score_cutoff                            100.0
  dn_pruning_conformer_score_scaling_factor                    2.0
  dn_pruning_clustering_cutoff                                 100.0
  dn_mol_wt_cutoff_type                                        soft
  dn_upper_constraint_mol_wt                                   550.0
  dn_lower_constraint_mol_wt                                   0.0
  dn_mol_wt_std_dev                                            35.0
  dn_constraint_rot_bon                                        15
  dn_constraint_formal_charge                                  2.0
  dn_heur_unmatched_num                                        1
  dn_heur_matched_rmsd                                         2.0
  dn_unique_anchors                                            1
  dn_max_grow_layers                                           9
  dn_max_root_size                                             25
  dn_max_layer_size                                            25
  dn_max_current_aps                                           5
  dn_max_scaffolds_per_layer                                   1
  dn_write_checkpoints                                         yes
  dn_write_prune_dump                                          no
  dn_write_orients                                             no
  dn_write_growth_trees                                        no
  dn_output_prefix                                             2ito_generic
  use_internal_energy                                          yes
  internal_energy_rep_exp                                      12
  internal_energy_cutoff                                       100.0
  use_database_filter                                          no
  orient_ligand                                                yes
  automated_matching                                           yes
  receptor_site_file                                           /gpfs/projects/AMS536/2024/students/group_1_2ITO/002_surface_spheres/2ITO.sph
  max_orientations                                             1000
  critical_points                                              no
  chemical_matching                                            no
  use_ligand_spheres                                           no
  bump_filter                                                  no
  score_molecules                                              yes
  contact_score_primary                                        no
  grid_score_primary                                           yes
  grid_score_rep_rad_scale                                     1
  grid_score_vdw_scale                                          1
  grid_score_es_scale                                           1
  grid_score_grid_prefix                                       /gpfs/projects/AMS536/2024/students/group_1_2ITO/003_gridbox/grid
  minimize_ligand                                              yes
  minimize_anchor                                              yes
  minimize_flexible_growth                                     yes
  use_advanced_simplex_parameters                              no
  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                                  250
  simplex_grow_tors_premin_iterations                          0
  simplex_random_seed                                          0
  simplex_restraint_min                                        no
  atom_model                                                   all
  vdw_defn_file                                                /gpfs/projects/AMS536/zzz.programs/dock6.10/parameters/vdw_de_novo.defn
  flex_defn_file                                               /gpfs/projects/AMS536/zzz.programs/dock6.10/parameters/flex.defn
  flex_drive_file                                              /gpfs/projects/AMS536/zzz.programs/dock6.10/parameters/flex_drive.tbl

Create a slurm file to run this:

  #!/bin/bash
  #SBATCH --job-name=2ito_generic
  #SBATCH --ntasks=28
  #SBATCH --nodes=1
  #SBATCH --time=48:00:00
  #SBATCH -p long-28core
  dock6 -i 2ito_generic.in

After the program has run, you can use the following command to see how many ligands has been generated. The program has made 368 potential ligands.

  grep MOLECULE 2ito_generic.denovo_build.mol2 | wc -l


The output file we are going to move to our local computer is 2ito_generic.denovo_build.mol2. We can see the ligands the program has created.

Go into Chimera and open the molecule on ViewDock. (Tools >> Surface/Binding Analysis >> ViewDock)

Use Column >> Show >> Grid_Score to find the ligand with the lowest energy. The ligand with the lowest score has a score of -93.318. 

[[File: 2ITO_genericDenovo_lowest_gridscore.png|thumb|center]]
[[File:2ITO_genericDeNovo_ligands_gridScores.png|thumb|center]]


We can compare this with the energy minimized ligand and see how it is positioned differently compared to the generated ligand (denovo_designed ligand is brown, energy minimized ligand is blue). This original energy minimized ligand has a grid score of -71.423.

[[File: 2ito_directory_structure.png|thumb|center]]
[[File: 2ito_directory_structure.png|thumb|center]]

We can also observe how the designed ligand and the original ligand interact with the receptor.

[[File: 2ito_directory_structure.png|thumb|center]]