Lead Optimization requires a detailed study and understanding of the binding modes of the receptor-ligand complexes. This knowledge will provide insight into which interactions a good inhibitor/antagonist has to make and ultimately lead to optimized drug compounds.
NOTE: binding modes of receptor-agonist and receptor-antagonist need not be exactly the same.

Once more we will use the estrogen alpha and beta nuclear receptor as working example. For the estrogen nuclear receptor, there are a number of crystal structures available in the Protein Data Bank:

Table 1: Some of the estrogen nuclear receptor crystal structures
PDB code	Name of Ligand	Type of Ligand
1A52	Estradiol	Agonist
3ERD	Diethylstilbestrol	Agonist
3ERT	4-hydroxytamoxifen	Antagonist
1ERR	Raloxifene	Antagonist

There are a number of ways to get information about the binding mode of a receptor without explicitly looking at the ligand-receptor complex with a molecular viewer:

Use PDBSUM to retrieve information on individual PDB entries. Especially interesting is the entry where you can get a plot of ligand interactions: Ligplot.

Use Ligand-Protein Contacts & Contacts of Structural Units to directly compare ligand contacts in agonist and antagonist structures.

Question:

Considering the contacts an agonist such as estradiol makes (1A52), how would you improve/change the antagonist 4-hydroxy-tamoxifen (3ERT) so that it can make an extra hydrogen bond ?

Hint: Use Ligand-Protein Contacts & Contacts of Structural Units to directly compare ligand contacts of the two crystal structures 1A52 and 3ERT.

You can also have a look at the Visualization of the ERα receptor chime page for a 3D representation of the differences between agonist and antagonist binding and the residues involved in ligand binding (Windows only).

Question:

Which crystal structure, 3ERT or 1A52, is best suited for antagonist-optimisation by docking and why ?

Now we are going to use Sybyl again to have a better look at the active site of the receptor.
(See the How to run Sybyl from your course account directory).

Read in the active site (3ERT):

File >> Read >> active_site.pdb >> OK >> No

File >> Molecular Spreadsheet >> New >> Database >> OK >> hits.mdb >> Open

Click on DIHYDROXY_TAMOXIFENE >> File (in the HITS windows) >> Put Rows into Molecular Area

Now lets give this ligand a distinct color:

View >> Color >> Atoms.. >> M2:DIHYDROXY_TAMOXIFENE >> All >> OK >> Orange >> OK

Lets add hydrogens to the active site:

Build/Edit >> Add >> Hydrogens >> M1:ACTIVE SITE >> OK

Remember a crystal structure does not contain hydrogens, so Sybyl has made a choice where the hydrogens are going to be attached. This is not always unambiguous. For our purpose the hydrogens on HIS524 are put in the wrong place. First label this histidine:

View >> Label >> Substructure >> M1:ACTIVE SITE >> Substructures >> HIS524 >> OK >> OK

Now lets tell sybyl to change this:

Biopolymer >> Composition >> Mutate Monomers >> M1:ACTIVE SITE >> Substructures >> HIS524 >> OK >> OK >> HIE >> OK

Now lets get back and add the hydrogens again (See above).
Lets change the orientation of the hydroxyl group on the Dihydroxytamoxifene, closest to the histidine.

But first we need the drawn hydrogen bonds to be updated dynamically:

View >> Display H-Bonds >> Dynamically

Chose the Rotatable Bonds icon from the vertical bar on the left of the screen (7th from the top, counting the tripos logo).

Click the leftmost field of the first free row.
Now click in the drawing screen (first mouse button) on the Oxygen of the hydroxyl and the Carbon that connects it in the ring.
You can change the orientation of the hydroxyl hydrogen by clicking and holding down the left and right arrows in the Rotatable Bonds window.
Use the arrows untill there is a hydrogen bond drawn.

Question:

Can you edit the ligand interactively to optimize the binding-mode ?

Lets pick an interaction with THR347, first label it (See above with the Histidine).
Now lets replace the hydrogen on the Dihydroxytamoxifene that is closest to this threonine with another group:

Build/Edit >> Add >> Group >> OH >> OK >> Replace >> Click on the hydrogen

Try to orient this new hydroxyl as we did before with the existing hydroxyl group. This is not so succesfull, but it turns out the OH group CAN make a hydrogen bond with a backbone carbonyl !!

Try other groups with the same procedure. (To return to the original situation delete atoms: Build/Edit >> Delete >> Atom >> M1:ACTIVE SITE >> click on the atom on the screen).

Also try the amide group.

Finally you can have a look at the following map of possible interaction sites in the active site of 3ERT:

The map with the hydrogens attached at the best positions of HIS524

The map is actually a .mol2 file. This can be read into sybyl, but first delete all other active molecules.

Build/Edit >> Zap (Delete) Molecule

You will also find these files in your directory ~/bioinf4/leadopt.

We have now provided the organic synthesists with new ligands to synthesize and test for biological activity.