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首页> 外文期刊>Chemistry: A European journal >In Situ Proteome Profiling and Bioimaging Applications of Small-Molecule Affinity-Based Probes Derived From DOT1L Inhibitors
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In Situ Proteome Profiling and Bioimaging Applications of Small-Molecule Affinity-Based Probes Derived From DOT1L Inhibitors

机译：DOT1L抑制剂衍生的基于小分子亲和力的探针的原位蛋白质组分析和生物成像应用

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摘要

DOT1L is the sole protein methyltransferase that methylates histone H3 on lysine 79 (H3K79), and is a promising drug target against cancers. Small-molecule inhibitors of DOT1L such as FED1 are potential anti-cancer agents and useful tools to investigate the biological roles of DOT1L in human diseases. FED1 showed excellent in vitro inhibitory activity against DOT1L, but its cellular effect was relatively poor. In this study, we designed and synthesized photo-reactive and "clickable" affinity-based probes (AfBPs), P1 and P2, which were cell-permeable and structural mimics of FED1. The binding and inhibitory effects of these two probes against DOT1L protein were extensively investigated in vitro and in live mammalian cells (in situ). The cellular uptake and sub-cellular localization properties of the probes were subsequently studied in live-cell imaging experiments, and our results revealed that, whereas both P1 and P2 readily entered mammalian cells, most of them were not able to reach the cell nucleus where functional DOT1L resides. This offers a plausible explanation for the poor cellular activity of FED1. Finally with P1/P2, large-scale cell-based proteome profiling, followed by quantitative LC-MS/MS, was carried out to identify potential cellular off-targets of FED1. Amongst the more than 100 candidate off-targets identified, NOP2 (a putative ribosomal RNA methyltransferase) was further confirmed to be likely a genuine off-target of FED1 by preliminary validation experiments including pull-down/Western blotting (PD/WB) and cellular thermal shift assay (CETSA).
机译：DOT1L是使赖氨酸79（H3K79）上的组蛋白H3甲基化的唯一蛋白质甲基转移酶，是一种有前景的抗癌药物。 DOT1L的小分子抑制剂（例如FED1）是潜在的抗癌药，也是研究DOT1L在人类疾病中的生物学作用的有用工具。 FED1对DOT1L表现出优异的体外抑制活性，但其细胞作用相对较差。在这项研究中，我们设计并合成了光反应性和“可点击的”基于亲和力的探针（AfBPs）P1和P2，它们是FED1的细胞渗透性和结构类似物。在体外和活的哺乳动物细胞（原位）中广泛研究了这两种探针对DOT1L蛋白的结合和抑制作用。随后在活细胞成像实验中研究了探针的细胞摄取和亚细胞定位特性，我们的结果表明，尽管P1和P2都容易进入哺乳动物细胞，但大多数都无法到达其中的细胞核。功能DOT1L驻留。这为FED1的细胞活性差提供了一个合理的解释。最后，使用P1 / P2进行大规模的基于蛋白质组的蛋白质组分析，然后进行定量LC-MS / MS，以鉴定FED1的潜在细胞脱靶目标。在鉴定出的100多个候选脱靶中，通过初步验证实验（包括下拉/蛋白质印迹法（PD / WB）和细胞）进一步证实NOP2（推定的核糖体RNA甲基转移酶）可能是FED1的真正脱靶。热位移分析（CETSA）。

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