17beta-Hydroxysteroid Dehydrogenase Type 14: Development of potent and selective inhibitors using crystallization-aided optimization and enzyme characterization

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17beta-Hydroxysteroid dehydrogenase type 14 (17beta-HSD14) is a recently characterized enzyme which is located in the brain, liver and placenta. The apoenzyme has been crystallized. It is able to convert both estradiol and dehydroepiandrosterone into estrone and 5-androstene-3beta, 17beta-diol, respectively but its functional role remains unclear. It has been suggested to be implicated in neuromodulation and in the pathogenesis of neurodegeneration. Potent and selective inhibitors are useful tools to study the role of an enzyme in a disease-oriented model but no inhibitor of this enzyme has been reported to date. In this project, we want to develop highly potent and selective inhibitors of 17beta-HSD14, to provide efficient tool compounds to investigate if this enzyme might be a good drug-target in neuronal diseases, to further structurally characterize the enzyme's active site and gain further understanding into the possible functional role of this enzyme. Starting from reasonably active 17beta-HSD14 inhibitors (identified in a preliminary study or by virtual screening), structural optimization will be performed using crystallography, modeling, chemical synthesis and biological evaluation in an iterative process. Analysis of the crystallized inhibitor-enzyme complex will provide information on the interactions achieved by the ligand with the enzyme and will structurally characterize the active site of the protein. In addition the 3D-structure will suggest other interactions between the enzyme and the inhibitor after structural modification of the latter. Modeling will validate the designed structures. After synthesis, 17beta-HSD14 potency will be evaluated in a NAD/NADH fluorescent-assay, which will be established in house and the potent compounds will enter a new cycle of optimization process. Selectivity toward functionally related enzymes like 17beta-HSD1, 2, 4 and 5 will be addressed using a structure-based or a ligand-based approach. Furthermore, screening for 17beta-HSD14 nonsteroidal substrates might provide an insight into a possible functional role of this enzyme.