Research interest in the development of histone deacetylase 8 (HDAC8) activators has substantially increased since loss-of-function HDAC8 mutations were found in patients with Cornelia de Lange syndrome (CdLS). A series of N-acetylthioureas (e.g., TM-2-51) have been identified as HDAC8-selective activators, among others; however, their activation mechanisms remain elusive. Herein, we performed molecular dynamics (MD) simulations and fragment-centric topographical mapping (FCTM) to investigate the mechanism of HDAC8 activation. Our results revealed that improper binding of the coumarin group of fluorescent substrates leads to the “flipping out” of catalytic residue Y306, which reduces the enzymatic activity of HDAC8 towards fluorescent substrates. A pocket between the coumarin group of the substrate and thed catalytic residue Y306 was filled with the activator TM-2-51, which not only enhanced binding between HDAC8 and the fluorescent substrate complex but also stabilized Y306 in a catalytically active conformation. Based on this newly proposed substrate-dependent activation mechanism, we performed structure-based virtual screening and successfully identified low-molecular-weight scaffolds as new HDAC8 activators.

自从在 Cornelia de Lange 综合征 (CdLS) 患者中发现 HDAC8 功能丧失突变以来，对组蛋白脱乙酰酶 8 (HDAC8) 激活剂开发的研究兴趣大幅增加。一系列N-乙酰硫脲（例如TM-2-51 ）已被鉴定为HDAC8选择性激活剂等；然而，它们的激活机制仍然难以捉摸。在这里，我们进行了分子动力学（MD）模拟和以片段为中心的拓扑图（FCTM）来研究HDAC8激活的机制。我们的结果表明，荧光底物香豆素基团的不当结合会导致催化残基 Y306 的“翻转”，从而降低 HDAC8 对荧光底物的酶活性。底物的香豆素基团和催化残基Y306之间的口袋被激活剂TM-2-51填充，这不仅增强了HDAC8与荧光底物复合物之间的结合，而且还稳定了Y306的催化活性构象。基于这种新提出的底物依赖性激活机制，我们进行了基于结构的虚拟筛选，并成功鉴定出低分子量支架作为新的HDAC8激活剂。