Insight II



6       Sketcher


Introduction

The Sketcher is accessed from the Builder module of Insight II. From the Sketch pulldown, choose the Toolbox command to bring up the MolBuilder. The power of the Sketcher is its ease of use and the speed with which complex structures can be built. You can draw a molecule essentially freehand, much like drawing on a piece of paper, and quickly convert the drawing into a reasonable 3D molecular structure. You can also use the Sketcher to build a structure in 3D.


Tutorial

This tutorial takes you through the process of building a structure in 2D, and in 3D. (It is numbered Application 2 because it is the second of 12 basic applications that give a general survey of Insight II functionality. To use the other applications start up Pilot from Insight II, then choose Insight II Tutorials.)


Application 2: Molecule Building

Building and Manipulating the ACE Inhibitor Captopril

Background

In the majority of cases in drug design the structure of the target enzyme or receptor is not known. The design of active compounds then relies upon the identification of functional groups that are critical for activity, and the definition of a 3D orientation of these groups that can be adopted in all active molecules: the so-called pharmacophoric pattern.

Angiotensin Converting Enzyme (ACE) is a zinc metallopeptidase which catalyzes the conversion of angiotensin I to angiotensin II, an octapeptide which is a potent vasoconstrictor. Compounds which inhibit ACE have an antihypertensive effect. Captopril (1-[(2S)-3-mercapto-2-methylpropionyl]-L-proline) (Figure 23) is a potent and specific ACE inhibitor and an orally effective antihypertensive agent (Hangauer 1989).

Extensive structure-activity studies have characterized the fundamental structural requirements for ACE inhibition as:

1.   a terminal carboxyl group which interacts with a positively charged residue in ACE;

2.   a carbonyl group involved in hydrogen bonding to a group in the active site;

3.   an effective zinc ligand, which in captopril is provided by the sulfhydryl group.

Using systematic conformational search techniques, a unique pharmacophoric model has been defined that was accessible by a series of 28 active and structurally diverse ACE inhibitors (Mayer et al. 1987, Dammkoehler et al. 1989). This pharmacophore is defined by a series of 5 interatomic distances between each of the groups known to be important in binding (see Figure 26).

Introduction

This application illustrates how to build the captopril molecule using the Sketcher. It goes on to demonstrate how to locate a conformation of captopril that is both low in energy (a conformation that on the basis of molecular mechanics energy calculations is energetically stable and attainable) and that also matches the pharmacophoric pattern. This is the "bioactive" conformation of captopril.

An important check of a pharmacophore model is that it should distinguish active from inactive compounds. A stereoisomer of captopril that has markedly reduced activity will also be built. This compound can only match the pharmacophore in a high energy (unstable) conformation. The inactivity of this compound may be explained by its inability to match the pharmacophore in a stable conformation.

 

Figure 23 . Captopril

 

Application

This application is organized into the following sections.

The structures that are to be built in this application have already been constructed should they be required:

Captopril_s.car initial 3D structure

Captopril_s.car2d 2D sketch

Captopril_active.car bioactive conformation

Captopril_r.car 3D structure of inactive isomer

Captopril_r.car2d 2D sketch of inactive isomer

If Insight II is running:

Enter delete * at the command prompt to remove any structures displayed in Insight II.

If Insight II is not running:

Enter biosym_tutorial at the UNIX prompt, and follow the on-screen
instructions.

Building Captopril in 3D

Using the Sketcher, 3D structures can be constructed by joining together units stored in a fragment library, and by a form of 3D sketching called Grow. The components of captopril are shown in chapter Figure 24 . Components of Captopril. Proline is a convenient fragment from which to start.

 

Figure 24 . Components of Captopril

 

1.   Go to the Builder module

Select Builder from the Module pulldown, by clicking the MSI logo. If the Molbuilder dialog box does not appear, select Molbuilder from the Toolbox pulldown menu.

2.   Access the necessary amino fragments

Open the Fragment Window by pressing the "3D Fragments..." button on the Molbuilder dialog box. When the window appears, and the fragments have been loaded, select Amino Acids from the pulldown menu labeled Fragment Libraries on the Fragment Window.

When accessing the fragment library for the first time in an Insight II session, data from the library is read in from disk. This takes a few seconds, after which the first amino acid fragments are displayed in the Fragment Window. Note that you may have more than one Fragment Library open at the same time.

Scroll through the fragments using the scrollbar at the bottom of the Fragment Window until Proline is one of the fragments displayed. Press the left mouse button down over any atom in Proline. When the cursor changes shape to a miniature drawing of the fragment, hold the left mouse button down while dragging the mouse into the main graphics window. This feature of the Fragment Window is called Drag and Drop. Use Object/Rename to change the name of the proline from PRO to captopril_s. Press Cancel.

The proline fragment is displayed in the graphics window, colored by atom. Note that you do not necessarily have to hold the mouse button over an atom to drag a fragment to the main graphics window. Whenever the cursor changes shape to respresent the fragment, the Drag and Drop feature is active.

CAPTOPRIL_S may be manipulated using the dials, or with the mouse. Orient CAPTOPRIL_S so that the NH group points to the left and the aldehyde group points down.

The complete CAPTOPRIL_S molecule is built by modifying this starting fragment.

3.   Add additional fragments

CAPTOPRIL_S currently exists as an object containing one residue--to ensure that further fragments are included as part of this residue, rather than as individual residues, turn on the Assimilate option.

Open the Defaults dialog box by selecting the "Defaults..." menu command in the Toolbox pulldown. Toggle the Assimilate Atoms default to On.

Note that, at this time, there are three open windows, the Molbuilder Dialog box, the Fragment Window, and the Defaults dialog box. All of these control windows can co-exist with the others, and any of the available controls can be used at any time.

To construct the amide bond an Aldehyde group is appended to the NH group. A fragment is added by selecting one hydrogen atom in the fragment and one hydrogen atom from the molecule to which the fragment is to be joined. Both hydrogen atoms are then deleted and a bond is created between the two heavy atoms to which the hydrogen atoms were bonded.

In the Fragment Window, press the "Fragment Libraries..." button. The Fragment Libraries dialog box allows opening and closing of multiple Fragment Libraries at a time. Turn Off the Common Fragments, turn On the Functional Groups libraries, and press OK. If the Aldehyde fragment isn't visible, scroll the window until it is. Pick either hydrogen atom in the Aldehyde fragment, then select the hydrogen bonded to the nitrogen in CAPTOPRIL_S and press the Single Bond button in the Molbuilder Dialog box.

The Aldehyde fragment is appended to CAPTOPRIL_S. Note that Insight II determined that the new bond is partially delocalized, and created a partial double bond. The background behind the Aldehyde fragment is now black, and a highlight should remain on the hydrogen that was selected. This is called the Hot Fragment. As long as a fragment is selected in this manner, you can bond the fragment to your molecule by selecting one or many hydrogens, and pressing the appropriate bond button.

If the "Add Torsions to New Bonds" button is turned On in the Defaults dialog box, a Torsion was also created on the new bond.

The value of the torsion is displayed at each bond. A cone on the Torsion shows which end will rotate (the larger end is the moving one). Clicking in empty space deactivates the Torsion (the cone disappears, and the font used for the value changes). To re-activate the Torsion, pick the bond again. To modify this torsion (only active Torsions may be modified), hold the middle mouse button down and move the mouse horizontally. To change the directionality of the Torsion (change the end that rotates), click on the cone.

There is evidence from both constrained analogs and conformational studies that the amide bond exists in the trans form in the bioactive conformation.

Select Molecule/Label, and set Label Property to Name. Pick CAPTOPRIL_S in the value aid. Press Cancel.

The geometry of the newly added aldehyde group is not quite right, yet. If there is no Torsion on the new bond (represented by a numerical value label on the bond), create one by picking on the center of the bond. Both atoms connected by the bond should be selected. Press the Torsion button on the main Icon Bar. Rotate the Torsion by moving the mouse left or right while holding the middle mouse button down. Change the geometry until the value becomes roughly 0.0, or the bond to the aldehyde hydrogen is trans to the N-CA bond. Click in empty space to de-activate the Torsion.

Drag and drop can also be used to create bonds to fragments.

Open the Fragment Libraries dialog box again, and turn Off Functional Groups, and turn On the Hydrocarbons library. Press the OK button. Press and hold the left mouse button over any hydrogen in the Ethyl fragment. Once again, the cursor will become a small drawing of the fragment. If the press was directly over a hydrogen atom, there will be a small square highlight over that atom in the cursor. With the mouse button held down, drag the cursor over the hydrogen remaining on the Aldehyde group just added to CAPTOPRIL_S, and release the mouse button. Press the Done button in the Fragment Window and the Defaults dialog box.

The Ethyl fragment is appended to the CAPTOPRIL_S.

Although the rest of the molecule could be built using fragments from the Fragment Library, it is faster to use the Grow functionality. At this time, only the Molbuilder dialog box should be open. The Grow mode uses the same controls that 2D sketching does in the Molbuilder dialog box, only they are used on 3D molecules.

Make sure no atoms are currently selected by clicking in empty space. Press either the Single Bond button, or the Draw button. Either of these controls activate the other, as well as the Carbon button. The cursor changes shape when moved over the graphics window to reflect the Element Type and the Bond Type selected.

Try selecting different bond types and element types to see the effect on the cursor shape. The cursor has these shapes only in the Draw mode, it is the usual Arrow shape while in Select mode.

Click on one of the hydrogens alpha to the carbonyl group. That hydrogen will become a methyl group, and if the Add Torsions to New Bonds button in the Defaults dialog box is turned On, a new Torsion is created as well.

In the Grow mode, Torsions are created dynamically. As you create new bonds, you can manipulate the geometry of the new Torsion. If you leave that Torsion active, the next Grow operation will replace it with a Torsion at the new bond. If you wish to have the Torsion remain, simply go to the Select mode and click in empty space to de-activate the Torsion. It will not be replaced in the next Grow operation.

The chirality at the new methyl group will be checked and if necessary, modified later. Atom types and bond orders are next modified to complete the CAPTOPRIL_S molecule.

4.   Add the sulfhydryl group

The sulfhydryl group can be added the same way.

Press the Sulfur button, leaving the Single Bond button selected. The cursor will reflect that you have Sulfur selected. Pick one of the terminal hydrogens on the ethyl fragment you previously added.

This hydrogen is converted to sulfur and the sulfur-carbon bond length is automatically adjusted.

5.   Create the carboxylate group

The only remaining task is to make the carboxylate group on the proline ring from the aldehyde. Although the hydrogen of the aldehyde group could be changed to an oxygen using the same method as the sulfur, we will use the Selection method to change both the element type and the two bond orders.

In Selection mode, the operating paradigm is "select, operate". The atoms that are to be operated on are selected, then the operation is choosen.

Go into the Selection mode by pressing the Select button. In this mode, atoms or bonds are selected, then the button representing the desired change is pressed. Select the aldehyde hydrogen, and press the Oxygen button.

This hydrogen is converted to oxygen and the oxygen-carbon bond length is automatically adjusted.

6.   Modify the bond orders

In Selection mode, any number of bonds or atoms can be operated on at the same time.

Select the two bonds in the carboxylate group either by clicking on the center of each bond, holding down the Shift Key while clicking on the second, or Box/Lasso select the three atoms. Then, press the Partial Double Bond button.

Both bond orders become partial double bonds. The model of CAPTOPRIL_S has been completed. Note that the labels on the heteroatoms do not appear by default as in the Figure below. They have been added to the graphic to clarify where those atoms are located. To create a display like the Figure below, first remove the current labels by choosing Label_action Off and selecting CAPTOPRIL_S as the Molecule Spec, then select each heteroatom individually (using the Shift key to add to the selection), go to the Label Molecule command and choose ATOM_SELECTION as the Molecule Spec, and Element as the Label Property.

The chirality at carbon atom D also needs to be checked. All chiral atoms in CAPTOPRIL_S can be labeled by chirality.

7.   Label all chiral atoms

Select Molecule/Label, and set Label Property to Chirality. Pick CAPTOPRIL_S in the value aid.

Since the default Molecule Pick Level is Molecule the atom selection defines the whole captopril molecule, and all chiral centers are labeled by chirality. In captopril, carbon D has chirality S.

If the chirality at carbon D is R, the chiral center must be inverted.

8.   Invert the chiral center, if necessary

Select "Stereochem..." from the Toolbox pulldown menu. The Stereochem dialog box. Box/lasso select the central chiral atom, and the two atoms adjacent (one is a hydrogen, and one is the methyl carbon). Press the Invert button on the Stereochem dialog box. Press Done to put away the dialog box.

CAPTOPRIL_S now has the correct stereochemistry. Before any further modeling it is important to refine the model in order to remove any bad atom-atom contacts created during the building process.

To remove the labels, toggle Label Option to Off, and select Execute. Press Cancel.

9.   Refine the model

You can use Optimize to refine the structure. The optimization is an iterative process which involves computing the energy of the structure using a molecular mechanics force field, calculating the forces acting on each atom and moving each atom to a new position in space in order to lower the forces (and thus reduce the energy). In order to calculate the energy, Insight II requires that the force field atom types (the potential types), and point charges be defined; as the structure has been built from various fragments the potential types and charges are not yet defined correctly. The Forcefield/Potentials command may be used to set potential types and charges, although in this instance this command does not need to be used as potential types and charges are set automatically when Optimize is invoked.

Press the Optimize button. The message "Now optimizing..." will appear in the Info Port, and the optimization process will start.

The optimization process starts immediately if there is only one molecule available in the graphics window. If there is more than one, you must select at least one atom in the molecule you want to optimize.

There are several adjustable parameters to the optimizer in the Molbuilder. Select the "Optimize_setup..." selection in the Toolbox pulldown menu. The controls to select the number of Iterations, the Derivative (Convergence criterion), and whether to use Partial Charges are available from this dialog box.

During the optimization, the energy, iteration number, and time used are reported at the top of the display. As the optimization proceeds the structure of CAPTOPRIL_S changes and the energy decreases. The optimization procedure automatically cycles through a number of optimization methods, commencing with a rapid method which is good at removing initial bad contacts. The optimization terminates with a very effective algorithm for refining small molecules that have been brought close to a minimum with the previous procedures.

10.   Save the refined structure

Select Molecule/Put. Ensure that the Assembly/Molecule parameter contains CAPTOPRIL_S, enter captopril_s for File Name, and select Execute. Press Cancel.

Note that naming of files (in contrast with the naming of objects) is case sensitive. CAPTOPRIL_S is now saved on disk in the Biosym format file captopril_s.car in the current directory. To retrieve this file use the Molecule/Get command, with Get File Type set to Archive.

Building Captopril in 2D

Sketching a molecule in two dimensions, followed by a conversion to three dimensions, is a convenient alternative to building in 3D. In many cases it is easier and quicker to build a molecule in this way.

Sketch does not impose any restrictions on how molecules are to be drawn; indeed they can be drawn with very poor geometry (e.g., very long bonds). The geometry of the sketch does not affect the quality of the three-dimensional structure, but it is recommended that sketches are kept chemically sensible.

 

Figure 25 . 2D Sketch of Captopril

  1.   If the Builder module pulldowns are not displayed, first go to Builder

Select Builder from the Module pulldown (i.e., the Biosym logo).

2.   Before starting the sketch, remove the CAPTOPRIL_S structure that was built with Builder

Type delete * on the command line, and press <Enter>.

3.   Begin sketching

Open the Defaults dialog box by selecting "Defaults..." in the Toolbox pulldown menu. In the "New Drawings are:" grouping, turn On the 2D Sketches button. Press Done. Press the 5-Membered Ring button in the Toolbox. As before in 3D, the Carbon button and the Draw Mode button will highlight. Double-click the left mouse button in the graphics window, and a cyclopentane will appear. Rotate the molecule so that one of the vertices points down.

You can also create a ring in the graphics window by clicking in one place, and drawing a line to another place. That vector will become one side of the ring.

The ring is displayed, and may be manipulated using the mouse. However, as it is a two-dimensional object, rotations around the x and y axes are not allowed. It is recommended that captopril is built in the orientation shown in chapter Figure 25 . 2D Sketch of Captopril.

4.   Sketch the side chain, initially as a four-carbon chain

Press the Single Bond button. As before, the Carbon and the Draw buttons highlight. Pick the starting atom (the atom on the left side of the ring--a beep indicates that an atom has been picked), move the mouse a bond length horizontally, and place the atom with a mouse click. Move the mouse again (a zig-zag style is recommend for chains) and place another atom. Repeat until the chain is 4 atoms long. Terminate the chain by clicking a second time on the last atom placed. A beep will also indicate that you have terminated the chain. Note that the rubber-band is not displayed when a chain is terminated.

5.   Draw the methyl group

Pick the second atom in the side chain, move the mouse a bond length vertically, place a new atom with a mouse click and terminate the chain with a second click.

6.   Add the carboxylate group

Pick the ring atom pointing down and move the mouse down a bond length and place the carbon atom. Press the Partial Double Bond button and the Oxygen button. Return to the display area and place the partially double bonded oxygen, and terminate the chain with a second click. Pick the carboxyl carbon, and place and terminate with another partially double bonded oxygen.

7.   Set the amide bond to partial double

Exit the Draw Mode by pressing the Select button. Pick the first bond of the sidechain (pick at the center of the bond--both atoms become highlighted). Then, press the Partial Double Bond button.

This operation could also have been done while in Draw mode.

8.   Draw the double bonded amide oxygen

Unselect the atoms by clicking in "empty space". Press the Double Bond button (this will, as before, automatically enter the Draw Mode), and the Oxygen button. Pick the first carbon in the side chain, move the mouse down a bond length. When you click for the second atom, the rubberband will terminate itself. This occurs when the valence of the added atom (the carbonyl oxygen) is completely satisfied.

9.   Create the correct element types

Change to the Select Mode, and select the ring atom connected to the sidechain,, and press the Nitrogen button. Lastly, select the atom at the end of the sidechain, and press the Sulfur button.

10.   Define stereochemistry

To complete the sketch, the stereochemistry needs to be defined. With the sketch in the orientation shown in chapter Figure 25 . 2D Sketch of Captopril, S chirality at both chiral centers is obtained using the Wedge Up Bond.

Unselect any atoms you may have selected by clicking in empty space. Press the Wedge_Up_Bond. Trace from the sidechain carbon to the methyl carbon. In the same way trace the bond from the ring to the carboxyl group as wedge up (pick the ring carbon first).

The sketch is now complete. Note that hydrogens will be added automatically in the 3D conversion process, so they are not necessary for sketches, except to specify stereochemistry.

11.   Save the sketch

Select Sketch/Put. Ensure that Sketch Name is set to SKETCH_01 (i.e. the first and only sketch in this session), and enter captopril_s as the File Name. Select Execute. Press Cancel.

The file captopril_s.car2d has been saved on disk in the current directory. It may be retrieved using the Sketch/Get command.

12.   Create the three-dimensional structure of captopril

Open the Defaults dialog box, and ensure that the Keep Sketch button in the Convert to 3D grouping is On. The state of the Check Valences button is unimportant at this time since the sketch we will convert has correct valences. To convert the sketch to a 3D structure, simply press the 2D->3D button. Press Done in the Defaults dialog box to put it away.

The 3D structure takes a few moments to generate. Although it is generated in a good conformation, it should be refined. Note that the potential types and charges are automatically set when the 3D structure was generated.

The Sketcher conversion automatically assigns a simple molecule name to the 3D molecule.

Use Object/Rename to change the name of the molecule from MOL_01 to captopril_s. Press Cancel.

13.  

Select an atom in the 3D molecule, and press the Optimize button.
Refine the 3D structure

Alternatively, you can select an atom of the 2D Sketch, and press the Optimize button. Insight II will first convert the sketch to 3D, then optimize the 3D molecule.

Watch the structure change and the energy decrease as the optimization proceeds. Before moving onto the next section, the refined 3D structure should be saved and the sketch deleted.

14.   Save the structure and delete the sketch

Select Molecule/Put. Ensure Assembly/Mol Level is set to Molecule level in the value aid, and the Assembly/Molecule parameter is set to CAPTOPRIL_S, enter captopril_sk_s for File Name, and select Execute. Press Cancel.

Select Object/Delete. Pick any atom in the 2D sketch and select Execute. Press Cancel.

The sketch is removed from the display (but note that it is still stored in the file created previously).

The Bioactive Conformation of Captopril

Whether built in 2D or 3D, the CAPTOPRIL_S structure is now displayed in the display area. This section illustrates the use of Torsions, Distance Monitors and Measure/Energy commands to locate the "bioactive" conformation of captopril.

The energy and specified interatomic distances are monitored interactively (using the Measure commands) while bond rotations are carried out using Torsions.

The ACE pharmacophore model is represented for captopril in chapter Figure 26 . The ACE Pharmacophore Model as a set of five interatomic distances between groups known to be important in binding.

 

Figure 26 . The ACE Pharmacophore Model


1 sulfur - carboxylate oxygen: 6.9 Å

2 sulfur - carbonyl oxygen 3.4 Å

3 carbonyl oxygen - carboxylate oxygen 3.8 Å

4 sulfur - carbonyl carbon 3.6 Å

5 carbonyl carbon - carboxylate oxygen 4.1 Å

1.   Monitor the energy of a molecule

Using the Measure/Energy command the van der Waals and Coulombic (electrostatic) energies of a molecule may be monitored interactively while the conformation is modified. Note that the energy reported will not be the same as that reported using Optimize, since Insight II only calculates the nonbonded interactions. When rotating around a bond the bond lengths and angles do not change, so their contributions to the energy do not need to be included.

Select Measure/Energy, and set Coulomb to Off. Ensure that Molecule Spec is set to CAPTOPRIL_S, and select Execute. Press Cancel.

The van der Waals, electrostatic (Coulombic), and total energy are reported at the top right of the display area. Note the energy. The Coulomb term is set off so the electrostatic energy is zero. ACE has a Zn2+ ion in the active site which would be expected to alter the charge distribution of any ligand, thus the charges that were automatically assigned to captopril during the optimization process may not be relevant.

2.   Define distance monitors

The conformation of the carboxylate group can be defined independently of the rest of captopril. The conformation of this group is determined by distances 3 and 5 which may be modified by rotating around Torsion 4 only, as the amide bond is known to be trans. If the amide bond is not currently trans, create a Torsion as before and rotate the bond to have a trans configuration. The conformation of the carboxylate group is set before moving on to the rest of the structure.

Select the atom pair defining distance 3 (the carbonyl oxygen to one carboxylate oxygen). Press the Measure button on the Icon Bar. Then select the atom pair defining distance 5 (the carbonyl carbon (C) to the same carboxylate oxygen), and press the Measure button again.

A distance monitor is displayed for each atom pair defined. The Measure Icon will create either Distance, Angle, or Dihedral Monitors depending on whether there are two, three, or four atoms selected. Unlike the menu-based Measure commands, the Measure Icon requires that for Angles and Dihedrals, the atoms are contiguous. The is because atom selections are not ordered, and thus non-contiguous ordering cannot be infered.

3.   Define the bond around which to rotate (Torsion 4 in chapter Figure 26 . The ACE Pharmacophore Model)

Select the two atoms that define torsion 4, (the ring carbon and the carbon of the carboxylate group), or select the bond between the two atoms. Press the Torsion button on the Icon Bar.
)

Press and hold down the middle mouse button while moving the mouse horizontally. The carboxylate group rotates, and the reported torsion angle changes.

Rotate the carboxylate group until the distance monitors match the required distances, the carbonyl oxygen-carboxylate oxygen distance is about 3.8, and the carbonyl carbon-carboxylate oxygen distance is about 4.1 Angstrom.

The energy has increased, but only slightly. As the conformation of the carboxylate group has now been defined, the distance monitors and torsion definition may be cleared.

4.   Clear monitors and de-activate the torsion

Click in `empty' space to de-activate the Torsion. The cone will disappear, and the value text change to a smaller font.

Both the Torsion and the Measure buttons are toggle buttons, and create or remove Torsions or measurements. To completely remove a Torsion, select the bond, or the two atoms, and press the Torsion button on the Icon Bar.

Select the carbonyl oxygen, and the oxygen of the carboxylate that had previously been selected for the Distance Monitor. Press the Measure button on the Icon Bar. Select the other two atoms involved in the remaining Distance Monitor, and press the Measure button again.

The distance monitors are removed.

5.   Modify the side chain conformation

In the same way the conformation of the side chain may be defined. The remaining 3 distance monitors (1, 2 & 4) are set up first.

First define Distance 1 between the sulfur and the carboxylate oxygen used in the previous measurements. Distance 2 is between the sulfur and the carbonyl oxygen, while Distance 4 is between the sulfur and the carbonyl carbon. Create each Distance Monitor by selecting each pair of atoms in succession, and pressing the Measure button.

Now define the 3 torsion angles 1, 2, and 3 in chapter Figure 26 . The ACE Pharmacophore Model.

Select the pair of atoms defining torsions 1 and press the Torsion button on the Icon Bar. Repeat this process for torsions 2 and 3.

Three torsions appear on the molecule. Note that at any one time, only one Torsion is active. You can iteratively rotate each Torsion by clicking on the bonds.

Iteratively manipulate torsions 1 and 2 to locate a low energy conformation in which the distance monitors match the pharmacophoric pattern.

Hint: In the "bioactive" conformation of captopril, torsion 1 has a value of 150 degrees. Rotate torsion 1 until the value reported is 150. Keeping torsion 1 at this value, rotate torsion 2 until the distances match the pharmacophore (chapter Figure 26 . The ACE Pharmacophore Model).

Rotating torsion 3 does not effect the distances, but once the correct conformation has been located rotating this torsion may lower the energy.

In the "bioactive" conformation, torsion 2 has a value of 90 degrees. Note that the energy has increased a little over the initial energy. This is not unexpected as the act of binding can induce strain in a ligand. With an energy increase of this size, this conformation of captopril will be energetically accessible.

6.   Clear the monitors and torsion definitions

In order to clear more than one Distance Monitor at a time, select Measure/Distance. Set Monitor Mode to Clear, and select Execute. Select Measure/Energy. Assure that Monitor is on and then set Monitor Mode to Clear. Select Execute.

The Energy Monitor text will no longer appear on the upper right of the graphics window.

Select Transform/Torsion. Set Torsion Operation to Clear. Select Execute.

7.   Save the bioactive conformation of CAPTOPRIL_S

Select Molecule/Put. Ensure Assembly/Molecule is set to CAPTOPRIL_S, enter captopril_active for File Name. Select Execute. Press Cancel.

The file captopril_active.car is stored on disk in the current directory in Biosym format. It may be retrieved using Molecule/Get.

The Inactive Stereoisomer

If the pharmacophore model is correct, it should not be possible for an inactive molecule to adopt the bioactive conformation without a significant cost in energy. The stereoisomer of captopril (1-[(2R)-3-mercapto-2-methylpropionyl]-L-proline) is markedly less active as an ACE inhibitor. Using the same techniques used above to locate the bioactive conformation of captopril, the ability of the inactive stereoisomer to adopt the bioactive conformation can be investigated.

The R stereoisomer can be built in one of two ways. If CAPTOPRIL_S was built using Sketch, the saved sketch can be read in using Sketch/Get, and modified by replacing the wedge up bond at the methyl group with a wedge down bond, followed by 3D conversion.

However, as the structure of CAPTOPRIL_S is present in the display area, it is simpler to invert the chirality at atom D (chapter Figure 26 . The ACE Pharmacophore Model).

1.   Modify the chirality

Open the Stereochem dialog box by selecting Stereochem... from the Toolbox pulldown menu. Select the methyl carbon, chiral atom D, and the bonded hydrogen, and press Invert. Press Done to put away the dialog box.

Captopril now has chirality R at this center. The molecule should be renamed:

Select Object/Rename. Pick any atom in CAPTOPRIL_S. Enter captopril_r as the New Name. Select Execute. Press Cancel.

This structure should be refined to remove the bad contacts generated as a result of the chirality inversion.

2.   Refine the structure

Select an atom in the molecule, and press the Optimize button.

The energy is initially high and drops rapidly as the optimization proceeds. (Check that the amide bond has not flipped into the cis conformation during the optimization. If it has, define a Torsion and rotate the bond to make it trans.)

With the procedures used in the previous section, attempt to locate a low energy conformation of CAPTOPRIL_R which matches the pharmacophore.

3.   Locate a conformation that matches the pharmacophore

First set the conformation of the carboxylate group by defining a Torsion to the carboxylate and Distance Monitors as before so that distances 3 and 5 match the pharmacophore.

Once the pharmacophoric conformation has been established, de-activate the Torsion, clear the Distance Monitors, and define the 3 side chain Torsions and 3 Distance Monitors. Use Measure/Energy to monitor the energy of the molecule. Set torsions 1 and 2 to those values of the bioactive conformation of CAPTOPRIL_S (150 and 90 degrees respectively).

In this conformation the distances match the pharmacophore quite well, but the energy is very high. Modifying the torsions locates lower energy conformations, but these do not match the pharmacophore. The inactivity of CAPTOPRIL_R may be rationalized by its inability to match the pharmacophore in a low energy conformation.

Discussion

This application has demonstrated how to use Insight II to build, modify, and refine a small molecule structure, and has illustrated the application of distance and energy monitors and bond rotations to locating a conformation which matches a known pharmacophoric pattern.

The captopril molecule used is simple enough to make it feasible to locate a conformation matching a known pharmacophore using these techniques. In most instances the structures in question will be more flexible and the pharmacophore will be undefined. In these situations the Search_Compare module may be used to perform rapid systematic conformational searches to locate conformations that match a set of distance constraints, and to generate and refine a pharmacophoric pattern for a series of active molecules.

Files Created

In addition to those files written using Molecule/Put and Sketch/Put, Insight II has automatically created a number of files, produced as a result of using Optimize. At this stage it is not important to understand the purpose of all of these files, but it is necessary to know the relevance of some of them.

The optimization of captopril_s produced files with the root name captopril_s0. The 0 is a counter (starting at zero) indicating that this was the first time Optimize was used in this session.

The initial coordinates of captopril_s (prior to optimization) are stored in captopril_s0.car, and the final optimized coordinates are stored in captopril_s0.cor. These files are both in the same format and may be read into Insight II using Molecule/Get, with Get File Type set to Archive. In addition to the coordinates these files contain the partial charges, atom names, and element types.

The .car and .cor are the only files produced by Optimize that can be read into Insight II. It is useful to have files that are created automatically, in order to back-track if a mistake is made at a later stage and the structure has not been saved using Molecule/Get.

The file captopril_s0.mdf is a definitions file, which along with other information contains the connectivity of the structure (i.e., which atom is bonded to which), and the potential type of each atom necessary for the molecular mechanics energy calculations.

The .inp file is the control file for the optimization, and the .out file lists the results of the optimization process.

A set of files with the same extensions was produced for the optimization of
captopril_r.




Last updated December 17, 1998 at 04:26PM PST.
Copyright © 1998, Molecular Simulations Inc. All rights reserved.