Continuing our theme of computational FBDD, in this paper Brožič et al. present their results of virtual screening using fragments. Aldo-ketoreductases control the activity of androgens, estrogens, and progesterone modulating the occupancy and transactivation of their receptors. Selectivity is key because the different AKR1C isoforms (1-4) have different physiological outcomes. Specifically, you want to inhibit 1 and 3, while having no activity at 2 and 4. Crystal structures exist for all four isoforms and there is a good history of drug discovery against these targets. Salicylates are selective for isoform 1, while phenylanthranilates are potent but unselective for all isoforms. 3-bromo-5-phenylsalicylic acid is 4.1 nM inhibitor against 1 and 20x selective against 2 and 100x for 3 and 4. Phenylaminobenzoates are potent and selective for 3.
A virtual screen followed by biochemical evaluation. Compounds from Asinex, Chembridge, Maybridge, and NCI were pulled from ZINC, yielding 1.9M cpds. After applying filters (fragment like properties, reactive groups, problematic groups, and predicted and known aggregators) they were left with 143000 cpds. Active site was defined as portion of the enzyme within 6 A of crystallized ligands. Docking (using FlexX) of these compounds in the active was performed with the mandated interaction that an H- bond acceptor no more than 3A from Y55 was present. 37 cpds for AKR1C1 and 33 for AKR1C3 were found. Of these 70 compounds, 11 were insoluble. Single point inhibition at 400 uM was performed on both enzymes, 1 and 3. Compounds with >55% Inh had the IC50 determined, as well as selectivity vs. 2. 25 actives were found against isoform 1 or 3, 11 of which are salicylates or aminobenzoates (known scaffolds) . Compounds 1-15 represent new chemical space (based upon similarity calculations) with known scaffolds against this slate of targets.
Compounds 16-28 represent structures from new chemical classes. 16-21 show no selectivity, 22 and 23 (ketone and aldehyde) show no selectivity and selectivity for Isoform 3 respectively. Compound 23 brings up the question, if you filtered to eliminate reactive groups how did an aldehyde make it through?
Compound 25 is selective for isoform 1, while 25-28 are selective for isoform 3. Compound 26 was the most interesting, as it was potent and selective for isoform 3 (based upon a fluorescence assay).
However, this selectivity could not be explained by docking (isoform 1 and 3 are 88% identical), which predicted the inhibition of isoform 1, but completely missed isoform 3 (docking rank 21509). The authors then did MD studies (10ns only) to see what could possibly happen. What follows is honest to goodness handwaving. What they conclude is that binding of 26 could induce conformational changes in both inhibitor and enzyme to make it bind really well. Isoform 3 has a larger SP1subpocket.
In a rare case of brutal (if unintended honesty), the authors state:
"It is obvious from these results that our virtual screening protocol is capable of finding potent AKR1C inhibitors but is unable to predict the isoform selectivity."