PO.CH01.05 · 化学
天然产物高通量筛选发现核糖体生物合成抑制剂
High-throughput screening of natural products discovers inhibitors of ribosome biogenesis
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
细胞必须在每个细胞周期使其核糖体丰度翻倍。因此，癌细胞对核糖体有很高的需求以匹配其高增殖速率。为此，核糖体生物合成已成为癌症干预的一个诱人靶点。作为进化军备竞赛的结果，自然界产生了丰富的生物活性分子来源，因此筛选天然产物馏分为发现靶向核糖体生物合成的新型化合物提供了机会。我们与NIH的国家推进转化科学中心（NCATS）合作，使用一种名为RiboSNAP的核糖体生物合成报告系统，筛选了NCI的天然产物文库以寻找核糖体生物合成抑制剂。RiboSNAP报告系统在内源性核糖体蛋白上整合了一个SNAP标签，可在活细胞中与荧光底物共价标记。随后可洗脱底物，留下新合成的SNAP标签蛋白，供后续用不同底物标记，以区分新旧核糖体。这使得能够追踪从核仁到细胞质新产生的核糖体，作为核糖体生物合成的读数。抑制剂发现基于以下发现：整个通路中核糖体生物合成的抑制会导致新翻译的SNAP标签RPL28蛋白滞留在核仁和/或细胞核中。我们小组及其他人此前的工作已确定，核仁形态可作为核糖体生物合成抑制剂作用机制的指标，因此不同的斑点形态提示对不同靶点的抑制。这种基于图像的分析表明，我们的筛选工作不仅识别出rRNA转录的抑制剂，还识别出rRNA加工和核糖体成熟的抑制剂。筛选完成后，我们的工作将转向纯化活性化合物和靶点鉴定，以加深我们对核糖体生物合成如何响应扰动的理解，并开发癌症干预措施。（本工作由5UG3CA290312-01资助）
查看英文原文 English abstract
Cells must double their ribosome abundance every cell cycle. Thus, cancer cells have a high demand for ribosomes to match their high proliferation rate. For this reason, ribosome biogenesis has become an alluring target for cancer interventions. As the result of an evolutionary arms race, nature has produced a rich source of bioactive molecules and thus screening natural product fractions presents an opportunity to discover novel compounds that target ribosome biogenesis. In collaboration with the NIH's National Center for Advancing Translational Science (NCATS) we have screened the NCI's natural products library for inhibitors of ribosome biogenesis using a ribosome biogenesis reporter system called RiboSNAP. The RiboSNAP reporter incorporates a SNAP tag on an endogenous ribosomal protein that can be covalently labeled with fluorescent substrates in live cells. Substrates can then be washed out leaving newly synthesized SNAP tagged protein free for subsequent labeling with different substrates to distinguish between old and new ribosomes. This allows for the tracking of newly produced ribosomes from the nucleolus to the cytoplasm as a readout for ribosome biogenesis. Inhibitor discovery is predicated on the finding that inhibition of ribosome biogenesis throughout the pathway results in retention of newly translated SNAP tagged RPL28 protein in the nucleolus and/or nucleus. Previous work by our group and others has determined that nucleolar morphology serves as an indicator of ribosome biogenesis inhibitor mechanism of action and therefore different puncta morphologies indicate the inhibition of different targets. This image-based analysis suggests our screening efforts have identified not only inhibitors of rRNA transcription, but also rRNA processing and ribosome maturation. Once screening is complete, our efforts will shift towards purification of the active compounds and target identification to further our understanding of how ribosome biogenesis responds to perturbations and to develop cancer interventions. (This work was supported by 5UG3CA290312-01)
利益披露 Disclosure
G. Kimble, None..
M. Jarajapu, None.