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

17 β-羟基类固醇脱氢酶14型（17 β-HSD 14）是一种新近鉴定的酶，其位于脑、肝和胎盘中。脱辅基酶已结晶。它能够将雌二醇和脱氢表雄酮分别转化为雌酮和5-雄烯-3 β，17 β-二醇，但其功能作用尚不清楚。它被认为与神经调节和神经变性的发病机制有关。有效的和选择性的抑制剂是研究酶在疾病导向模型中的作用的有用工具，但迄今为止还没有这种酶的抑制剂的报道。在这个项目中，我们希望开发高效和选择性的17 β-HSD 14抑制剂，以提供有效的工具化合物来研究这种酶是否可能成为神经元疾病的良好药物靶点，进一步结构表征酶的活性位点，并进一步了解这种酶可能的功能作用。从具有合理活性的17 β-HSD 14抑制剂（在初步研究中或通过虚拟筛选确定）开始，将在迭代过程中使用晶体学、建模、化学合成和生物学评价进行结构优化。对结晶的配体-酶复合物的分析将提供关于配体与酶实现的相互作用的信息，并将在结构上表征蛋白质的活性位点。此外，3D结构将表明酶和抑制剂之间的其他相互作用后，后者的结构修饰。建模将验证所设计的结构。合成后，将在NAD/NADH荧光测定中评价17 β-HSD 14的效力，该测定将在内部建立，有效化合物将进入新的优化过程循环。对功能相关酶如17 β-HSD 1、2、4和5的选择性将使用基于结构或基于配体的方法来解决。此外，筛选17 β-HSD 14非甾体底物可能提供了一个深入了解这种酶的可能的功能作用。

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.