Institute of Pharmacy, Martin-Luther University of Halle-Wittenberg, Wolfgang-Langenbeck-Str. 2-4, 06120, Halle/Saale, Germany.
Institute of Toxicology, Johannes-Gutenberg University of Mainz, Obere Zahlbacher Str. 67, 55131, Mainz, Germany.
Eur J Med Chem. 2022 Apr 15;234:114272. doi: 10.1016/j.ejmech.2022.114272. Epub 2022 Mar 11.
Histone deacetylases (HDACs) are a family of 18 epigenetic modifiers that fall into 4 classes. Histone deacetylase inhibitors (HDACi) are valid tools to assess HDAC functions. HDAC6 and HDAC10 belong to the class IIb subgroup of the HDAC family. The targets and biological functions of HDAC10 are ill-defined. This lack of knowledge is due to a lack of specific and potent HDAC10 inhibitors with cellular activity. Here, we have synthesized and characterized piperidine-4-acrylhydroxamates as potent and highly selective inhibitors of HDAC10. This was achieved by targeting the acidic gatekeeper residue Glu274 of HDAC10 with a basic piperidine moiety that mimics the interaction of the polyamine substrate of HDAC10. We have confirmed the binding modes of selected inhibitors using X-ray crystallography. Promising candidates were selected based on their specificity by in vitro profiling using recombinant HDACs. The most promising HDAC10 inhibitors 10c and 13b were tested for specificity in acute myeloid leukemia (AML) cells with the FLT3-ITD oncogene. By immunoblot experiments we assessed the hyperacetylation of histones and tubulin-α, which are class I and HDAC6 substrates, respectively. As validated test for HDAC10 inhibition we used flow cytometry assessing autolysosome formation in neuroblastoma and AML cells. We demonstrate that 10c and 13b inhibit HDAC10 with high specificity over HDAC6 and with no significant impact on class I HDACs. The accumulation of autolysosomes is not a consequence of apoptosis and 10c and 13b are not toxic for normal human kidney cells. These data show that 10c and 13b are nanomolar inhibitors of HDAC10 with high specificity. Thus, our new HDAC10 inhibitors are tools to identify the downstream targets and functions of HDAC10 in cells.
组蛋白去乙酰化酶（HDACs）是一个 18 种表观遗传修饰物家族，分为 4 个类。组蛋白去乙酰化酶抑制剂（HDACi）是评估 HDAC 功能的有效工具。HDAC6 和 HDAC10 属于 HDAC 家族的 IIb 亚类。HDAC10 的靶点和生物学功能尚未确定。造成这种情况的原因是缺乏具有细胞活性的特异性和有效的 HDAC10 抑制剂。在这里，我们合成并表征了哌啶-4-丙烯羟肟酸作为高效且高选择性的 HDAC10 抑制剂。这是通过将基本的哌啶部分靶向 HDAC10 的酸性门控残基 Glu274 来实现的，该部分模拟了 HDAC10 的多胺底物的相互作用。我们通过 X 射线晶体学证实了所选抑制剂的结合模式。根据使用重组 HDACs 进行的体外分析，基于其特异性选择了有前途的候选药物。最有前途的 HDAC10 抑制剂 10c 和 13b 与 FLT3-ITD 癌基因一起在急性髓系白血病（AML）细胞中进行了特异性测试。通过免疫印迹实验，我们评估了组蛋白和微管蛋白-α的乙酰化，它们分别是 I 类和 HDAC6 的底物。作为 HDAC10 抑制的验证试验，我们使用流式细胞术评估神经母细胞瘤和 AML 细胞中自噬溶酶体的形成。我们证明 10c 和 13b 高度特异性地抑制 HDAC10，而对 HDAC6 没有显著影响，并且对 I 类 HDAC 没有显著影响。自噬溶酶体的积累不是细胞凋亡的结果，并且 10c 和 13b 对正常人肾细胞没有毒性。这些数据表明，10c 和 13b 是具有高特异性的 HDAC10 的纳摩尔抑制剂。因此，我们的新型 HDAC10 抑制剂是识别细胞中 HDAC10 的下游靶点和功能的工具。
