DOT1L (the disruptor of telomeric silencing 1-like), through its methyltransferase activity of H3K79, plays essential roles in transcriptional regulation, cell cycle regulation, and DNA damage response. In addition, DOT1L is believed to be involved in the development of MLL-rearranged leukemia driven by the MLL (mixed-lineage leukemia) fusion proteins, which thus to be a crucial target for leukemia therapy. Hence, discovering of novel DOT1L inhibitors has been in a great demand. In this study, we initiated the discovering process from setting up the AlphaLISA based High Throughput Screening (HTS) assay of DOT1L. Combining with radioactive inhibition assay and Surface Plasmon Resonance (SPR) binding assay, we identified compound 3 and its active analogues as novel DOT1L inhibitors with IC₅₀ values range from 7 μM to 20 μM in vitro. Together with the analysis of structure activity relationships (SAR) and binding modes of these compounds, we provided clues to assist in the future development of more potent DOT1L inhibitors. Moreover, compounds 3 and 9 effectively inhibited the proliferation of MLL-rearranged leukemia cells MV4-11, which could induce cell cycle arrest and apoptosis. In conclusion, we developed a HTS platform based on AlphaLISA method for screening and discovery of DOT1L novel inhibitor, through which we discovered compound 3 and its analogues as potent DOT1L inhibitors with promising MLL-rearranged leukemia therapeutic application.

DOT1L（端粒沉默1 -like的破坏者）通过其H3K79的甲基转移酶活性，在转录调控，细胞周期调控和DNA损伤反应中起着至关重要的作用。此外，据信DOT1L参与了由MLL（混合谱系白血病）融合蛋白驱动的MLL重排白血病的发展，因此，它是白血病治疗的关键靶标。因此，迫切需要发现新型的DOT1L抑制剂。在这项研究中，我们从建立基于AlphaLISA的DOT1L的高通量筛选（HTS）检测方法开始了发现过程。结合放射性抑制测定和表面等离子体共振（SPR）结合测定，我们确定了化合物3及其活性类似物为具有IC _50的新型DOT1L抑制剂体外值从7μM到20μM不等。连同对这些化合物的结构活性关系（SAR）和结合模式的分析一起，我们提供了线索，以协助将来开发更有效的DOT1L抑制剂。而且，化合物3和9有效抑制MLL重排的白血病细胞MV4-11的增殖，这可以诱导细胞周期停滞和凋亡。总之，我们开发了一种基于AlphaLISA方法的HTS平台，用于筛选和发现DOT1L新型抑制剂，通过该平台我们发现了化合物3及其类似物作为有力的DOT1L抑制剂，有望在MLL重排的白血病治疗中得到应用。