Pharmaceutical Chemistry, Philipps University Marburg, Marbacher Weg 6, 35037 Marburg, Germany.
Helmholtz Zentrum München, Research Unit Molecular Endocrinology and Metabolism, 85764 Neuherberg, Germany.
J Steroid Biochem Mol Biol. 2021 Feb;206:105790. doi: 10.1016/j.jsbmb.2020.105790. Epub 2020 Nov 24.
17β-Hydroxysteroid dehydrogenase type 2 (17β-HSD2) catalyzes the conversion of highly active estrogens and androgens into their less active forms using NAD as cofactor. Substrate and cofactor specificities of 17β-HSD2 have been reported and potent 17β-HSD2 inhibitors have been discovered in a ligand-based approach. However, the molecular basis and the amino acids involved in the enzymatic functionality are poorly understood, as no crystal structure of the membrane-associated 17β-HSD2 exists. The functional properties of only few amino acids are known. The lack of topological information impedes structure-based drug design studies and limits the design of biochemical experiments. The aim of this work was the determination of the 17β-HSD2 topology. For this, the first homology model of 17β-HSD2 in complex with NAD and 17β-estradiol was built, using a multi-fragment "patchwork" approach. To confirm the quality of the model, fifteen selected amino acids were exchanged one by one using site directed mutagenesis. The mutants' functional behavior demonstrated that the generated model was of very good quality and allowed the identification of several key amino acids involved in either ligand or internal structure stabilization. The final model is an optimal basis for further experiments like, for example, lead optimization.
17β-羟类固醇脱氢酶 2 型（17β-HSD2）利用 NAD 作为辅助因子将高度活跃的雌激素和雄激素转化为其不活跃的形式。已经报道了 17β-HSD2 的底物和辅助因子特异性，并且已经在基于配体的方法中发现了有效的 17β-HSD2 抑制剂。然而，由于没有膜相关 17β-HSD2 的晶体结构，因此对酶功能的分子基础和涉及的氨基酸知之甚少。只有少数氨基酸的功能特性是已知的。缺乏拓扑信息阻碍了基于结构的药物设计研究，并限制了生化实验的设计。这项工作的目的是确定 17β-HSD2 的拓扑结构。为此，使用多片段“拼凑”方法，构建了与 NAD 和 17β-雌二醇结合的 17β-HSD2 的第一个同源模型。为了确认模型的质量，使用定点突变技术逐个交换了十五个选定的氨基酸。突变体的功能行为表明，生成的模型质量非常好，并确定了几个涉及配体或内部结构稳定的关键氨基酸。最终模型是进一步实验的理想基础，例如先导优化。
