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.

组蛋白脱乙酰酶 (HDAC) 是一个由 18 种表观遗传修饰剂组成的家族，可分为 4 类。组蛋白脱乙酰酶抑制剂 (HDACi) 是评估 HDAC 功能的有效工具。HDAC6 和 HDAC10 属于 HDAC 家族的 IIb 类亚群。HDAC10 的目标和生物学功能不明确。这种知识的缺乏是由于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-丙烯酰异羟肟酸盐作为 HDAC10 的有效和高选择性抑制剂。这是通过用模拟 HDAC10 多胺底物相互作用的碱性哌啶部分靶向 HDAC10 的酸性看门人残基 Glu274 来实现的。我们已经使用 X 射线晶体学确认了选定抑制剂的结合模式。通过使用重组 HDAC 进行体外分析，根据它们的特异性选择有希望的候选者。最有前途的 HDAC10 抑制剂测试了10c和13b在具有 FLT3-ITD 致癌基因的急性髓性白血病 (AML) 细胞中的特异性。通过免疫印迹实验，我们评估了组蛋白和微管蛋白-α 的过度乙酰化，它们分别是 I 类和 HDAC6 底物。作为 HDAC10 抑制的有效测试，我们使用流式细胞术评估神经母细胞瘤和 AML 细胞中自溶酶体的形成。我们证明10c和13b抑制 HDAC10 比 HDAC6 具有高特异性，并且对 I 类 HDAC 没有显着影响。自溶酶体的积累不是细胞凋亡的结果，10c和13b对正常人肾细胞没有毒性。这些数据表明10c和13b是具有高特异性的纳摩尔级 HDAC10 抑制剂。因此，我们的新 HDAC10 抑制剂是识别细胞中 HDAC10 下游靶标和功能的工具。