HINT 2.30 Manual: Chaper 6S

LESSON 5: InterMolecular Table and Map Calculations

This lesson assumes that you have just completed Lesson 4. If you have, delete the backgrounds created in Lesson 4 and go to Step 2 of this lesson. Otherwise, follow the instructions in Step 1 of Lesson 1 and Steps 1, 2 and 3 of Lesson 3.
  1. Read in the inhibitor

    Read in the inhibitor A74704. Go to the File pulldown and select the Read... command. Choose a74704.pdb as the input file; press OK. Be sure to choose No when asked to Center the Molecule.

  2. Prepare the inhibitor for HINT calculations

    Because this molecule was read into HINT as a PDB file, you will have to "clean it up" by adding appropriate bonds and SYBYL atom types before HINT can Partition or otherwise perform calculations on it. To give us working space, first "blank" HIV1 (M1) by using the Check Box icon under the Tripos logo. Now color the atoms of A74704 by their type (View, Color, i By Type, M2) and label the atoms by their type (View, Label, Atom Type..., M2, All) to make it easier for us to clean up A74704.

    What you need to do is ensure that the correct bond order is set for the atoms in the A74704 inhibitor. You probably note immediately that the four phenyl rings do not have the expected partial double bonds. Less evident but equally problematic is that there are two missing and one spurious bond. First we will correct the latter problem. Note that SYBYL chose to draw a bond between two of the carbonyl carbons, instead of the expected amide bonds between these carbons and the amide nitrogens. Delete the spurious bond with (Build/Edit, Delete, Bond...) by picking the atoms at each end of the bond. Now add the appropriate new bonds (Build/Edit, Add, Bond...) by picking the atoms between which you want to build the bond. Also note that one of the connected nitrogens has an incorrect atom type: instead of N.am it is labeled as N.2. Now we will change all incorrect atom types: (Build/Edit, Modify, Atom, TYPE); trace around all four of the phenyl rings by selecting each atoms and also pick the incorrectly typed nitrogen. When you press OK you will be prompted one-by-one for the new Atom type of each atom; choose C.ar for all of the carbons and N.am for the nitrogen. At completion the atom types will change, and the bonds will change to aromatic display.

    Corrected A74704 model

    Return the HIV1 (M1) molecule display by using the Check Box icon.
  3. Partition the A74704 inhibitor and Re-partition HIV1

    Select Molecule from the HINT Partition submenu. Pick All atoms of M2 (A74704) and the Partition Method should be Calculate. Use Hydrogen Treatment of Essential and Via Bonds Polar Proximity. Press OK. HINT calculates the LogP for A74704 to be 6.42.

    The HIV1 molecule was previously partitioned with Directional Vectors. We must re-partition with this feature set to Spherical. Select Molecule from the HINT Partition submenu. Pick All atoms of M1 (HIV1) and the Partition Method should be Dictionary. Use Hydrogen Treatment of Essential, Solvent Condition set to Neutral and Via Bonds Polar Proximity. Direction Vectors is on, click on Parameters and select Spherical. Press OK and OK. The LogP value will again be -9.00.

  4. The InterMolecular HintTable Calculation for A74704 and HIV1

    Call the HintTable InterMolecular dialog box. In this box set the parameters as follows (Interactions: All, Cut Off Radius: 6.00, Van der Waals Limit: 1.00, Output Radius: 6.00, Output Value: 10.00, Proton Suppress: on, H-Bnd Distance X-Y: 3.65). These last three parameters setup the calculation to create an interaction list that contains all interactions within 6.00 Angstroms, having an intensity (MicroInteraction Constant) of 10.00 or greater, and defining a hydrogen bond as an interaction between H-bond donor and H-bond acceptor atoms of 3.65 Angstroms or less. Choose Molecule... to be M1 (HIV1) and Molecule2... to be M2 (A74704). In the Distance Function dialog box set the parameters as follows (Hydropathic Term: exp(-nr), Steric term: Lennard_Jones6-12, Steric/Hydro Scaler: 50. Enter hiv1_a74704_in.tab as the Table File name. Press OK when all these are set to begin the HintTable calculation. The resulting Total Interaction Constant (882) represents a prediction from HINT of the binding efficiency of this inhibitor in HIV1. In the absence of other information, this single number is more or less meaningless. However, correlation of Hint Interaction Constant predictions with experimental binding data for several compounds may lead to a model that can be used with new, untested molecular inhibitors. This InterMolecular HintTable calculation also creates the ASCII text file hiv1_a74704_in.tab in the current directory. You can examine this file at any time by opening a new unix window and listing or editing the file directly under unix. It may also be a good idea to print this file. In a later step of this lesson we will examine these HintTable results concurrent with results of a HintMap calculation.

    HINT Interaction Table for HIV-1 and A74704

  5. The InterMolecular HintMap Calculation for A74704 and HIV1

    In this step you will construct 3-D maps that enable visualization of the specific binding interactions between the A74704 inhibitor and HIV1. Select the HintMap InterMolecular command from the Hint submenu (under eslc on the SYBYL menubar). First, describe the grid region for the calculation by pressing the Region Definition... button. Use Molecule Extents where Molecule... is M2 (A74704). This defines a region around the active site as discussed in Lesson 4. Also set a Margin of 4 and a Grid Resolution of 1.25) You will perform the calculation twice so that hydrophobic binding interactions can be distinguished from polar binding interactions. First, for the hydrophobic interactions, set Interactions: Hydrophobic, Cut Off Radius: 8.00, Van der Waals Limit: 1.00. In the Distance Function... dialog set Hydropathic Term: exp(-nr), Steric term: Lennard-Jones6-12, and Steric/Hydro Scaler: 50. Select Molecule... to M1 (HIV1) and Molecule2... to M2 (A74704). This calculation will take 20-30 minutes, so run it in Batch. Set Map File name as hiv1_a747_in_h.cnt. Press OK to launch SYBYL NetBatch.

    Next, for the polar interactions, set all parameters the same as above except: 1) Interactions: Polar; 2) Map File name: hiv1_a747_in_p.cnt; 3) Job Name: HintInterMap2.

    After both calculations have completed, the resulting HintMaps can be contoured and displayed (HintMap Contour). Contour the hydrophobic interaction map (hiv1_a747_in_h.cnt) with +400 (green). Contour the polar interaction map (hiv1_a747_in_p.cnt) with -800 (red) and +800 (blue). These contours represent the significant interactions between the A74704 inhibitor and HIV1. The green regions represent hydrophobic/hydrophobic interactions that promote the binding; the blue regions represent polar/polar interactions such as acid/base, hydrogen bonding, Coulombic, etc. that promote the binding; and the red regions represent polar/polar interactions that appear to discourage the inhibitor binding. Numerical output from HintTable calculations aid in the interpretation of the Interaction HintMaps. There is also significant information in the negative portion of the hydrophobic interaction map (contour hiv1_a747_in_h.cnt at -400, color purple) -- these data represent hydrophobic/polar interactions that appear to discourage binding. We advise you treat these data with care, the presence of these negative interactions is an inescapable consequence of biological interactions, and it may be tempting to extract too much significance from them.

    HINT Interaction map for HIV-1 and inhibitor A74704

  6. Modify Protein Model to Improve Binding

    One interesting observation in the HIV1/A74704 system is the crucial interaction between the core hydroxyl of the substrate and the carboxylates of ASP 25 and ASP 125. Close examination of this region suggests that a better protein model may be obtained by protonating one of the two ASP residues to add a second hydrogen bond between the substrate and HIV1 in this region. Add a proton to the OD2 atom of ASP 25. The easiest way to do this in SYBYL is to first Color and Label the Atoms by Name in the ASP25 substructure by using commands in the View pulldown on the menubar. To add a hydrogen to OD2> we will first have to change the atom types of OD1 and OD2 to reflect the new bonding pattern. From Build/Edit, Modify the Atom... ONLY_TYPE of OD1 to O.2 and OD2 to O.3. Add an Atom (Build/Edit) to OD2 (type H). Now the new hydrogen will have to be named (Build/Edit, Modify, Atom..., NAME) with the QUERY mode. The new name should be HD2. Modify the torsion around the new O-H bond until the minimum distance between the new hydrogen and the substrate hydroxy oxygen (303:OX) (ca. 2.27 Angstrom) is reached. Do this with the Torsion (Rotatable Bond) icon under the Tripos logo. Pick the first blank box to define the rotatable bond. (Pick CG and OD2 on the ASP25 substructure.) Then set up distance monitoring (View, Monitor, Distance..., ADD) between HD2 (ASP25) and OX (A74704). Adjust the torsion with the arrows in the Rotatable Bonds gadget.

    Repeat the HintTable calculation from the beginning, remembering to Partition HIV1 with the Inferred Solvent Condition so that the modified ASP 25 is properly registered. The HINT interaction constant should now be about 1660 representing an improvement of about 40 % over the original model.