Two hydrophobic features were created at the junctions of rings A and B and of rings C and D by selecting two ring annotation points at a time and then pressing Feature in the Query Editor. Feature F2, where the interaction with HIS221 takes place, was edited to be an acceptor instead of a donor-acceptor. Both F1 and F2 were marked as being essential.
To construct a query that is selective for flavonoids, we began with flavone (note, however, that four of the flavonoids in the test database were isoflavones). From the four pharmacophoric annotation points, three were chosen to construct a 3-point pharmacophore, as illustrated below.
This query differs from the estradiol query in several significant ways. Although the two aromatic features overlap the hydrophobic features of the estradiol query, they are smaller, and both are specifically aromatic. Also, the hydrogen bond site features of estradiol were not included in the initial flavone query; instead, an acceptor at the carbonyl oxygen was used.
After generating an initial query, the test database was searched. The results of the search were then examined to suggest modifications to the query.
Prior to searching, pharmacophore annotations were calculated for all molecules in the test database using the Pharmacophore Preprocessor, a utility that is also integrated into the Pharmacophore Search application. This step was done to allow for faster searching.
In the Pharmacophore Search panel, Hit Entries were specified to be selected in the Database Viewer, in addition to being written out to a separate results database. Using the estradiol pharmacophore from above resulted in 4 hit molecules, of which 3 were steroids (including estradiol), and 1 was a flavonoid.
The small hit set suggested that the query might be overconstrained. When a partial match of a minimum of 3 features was permitted, the result was 14 hit molecules, of which 3 were steroidal and 7, flavonoid. The hit set included both estradiol and equilin, as well as coumesterol which, although non-steroidic, is similar to estradiol in structure. About 1/3 of the compounds had good to moderate activity.
Quick experimentation using the Ignore flag in the Pharmacophore Query Editor showed that hydrophobic feature F3 could be eliminated from the query without affecting results.
When this query was applied to the search database, the hit set included most of the steroidal compounds in the database.
With the flavone pharmacophore query, 20 hit molecules were obtained, of which 1 was non-flavonoid (coumesterol), and 19 were flavonoids; none were steroids. Formonetin and daidzein, both isoflavones, were missed by the query. Note, however, that genistein and biochanin A, also isoflavones, were successfully matched. An examination of the output hit conformer database confirmed that the presence of extra hydroxyl groups in these latter compounds permit a good superposition with the pharmacophore query, as shown below for genistein (magenta), superimposed upon flavone (blue). This result underlies the importance of close examination of pharmacophore search results, and may even suggest alternate ligand binding modes.
Note that preference for flavones over isoflavones may in fact be desirable since isoflavones appear to exhibit greater estrogenicity than flavones.
Genistein (magenta) Superimposed upon Flavone (blue)
When this flavonoid query was applied to the search database, 350 hits were obtained, or slightly over 18% of the compounds, none of which were steroidal in nature.
The flavone query is missing the two important hydrogen bonding sites observed in the native bonding interaction of 1FDT. Adding these features to the query may help enforce a better binding position of the candidate ligands. To recover these features, we loaded flavone along with estradiol, apigenin, and coumesterol molecules into MOE. These latter three ligands all exhibit hydrogen bonding contact with TYR155 and HIS221. Three molecules were used preferentially over a single molecule to include some variability in positioning. Using the Pharmacophore Consensus application, we obtained a consensus query. The Pharmacophore Consensus application calculates a query composed of all features of all molecules, with an indication of the proportion of shared features.
We selected the two hydrogen bonding sites, G3 and G4, to be added to the original flavone query. Then, the constraints on the query were relaxed slightly to allow a partial match of a minimum of four features, to reflect the variability in the test ligands. Finally, we relaxed the aromatic condition on the two aromatic features, allowing a non-aromatic hydrophobic feature to also match at those points (the feature type was set to Aro|Hyd).
Flavone (magenta), Estradiol (blue), Apigenin (red), and Coumesterol (green) with New Query
A search of the test database using this new consensus query yielded 17 hits, including 15 flavonoids, 1 non-flavonoid (coumesterol), and 1 steroid (estradiol). The hit set included those test compounds having highest inhibitory activity, including coumesterol, apigenin, apigenin analogs, and genistein.
The query was then applied to the search database compounds, in which case 201 hits were obtained, or about 10.5% of the compounds.
A visual inspection of the output of the search revealed molecules that should be ruled out for steric reasons. An additional volume constraint was therefore added to the query to help further constrain the search. Using the Union feature of the Pharmacophore Query Editor, excluded volume spheres were positioned coincident with atoms in the active site of the 1FDT complex that were within 5 A of the bound ligand. The radii of the spheres was set to 1.4 A.
Cutaway View of 1FDT Pocket with Query Containing Excluded Volumes
Using the supplemented query on the search database yielded 134 hits, or about 7% of the molecules in the database.
3D pharmacophore generation and searching is an important technique used in the identification of candidate active ligands. In MOE, 3D queries can contain locations of pharmacophore features or chemical groups as well as restrictions on shape imposed by specifying included and/or excluded volumes. An interactive editor allows for both query customization and elucidation of a consensus query from a set of aligned molecules. Such a query can then be used to filter a conformation database. The pharmacophoric database search application provides a high degree of control, offering both partial and systematic matching as well as flexible matching rules. MOE's pharmacophore tools are integrated together, making the process of iterative pharmacophore model generation and refinement easier.
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