Cautain Bastien, de Pedro Nuria, Murillo Garzón Virginia, Muñoz de Escalona María, González Menéndez Victor, Tormo José R, Martin Jesús, El Aouad Noureddine, Reyes Fernando, Asensio Francisco, Genilloud Olga, Vicente Francisca, Link Wolfgang
1Fundación MEDINA, Parque Tecnológico Ciencias de la Salud, Granada, España.
J Biomol Screen. 2014 Jan;19(1):57-65. doi: 10.1177/1087057113501389. Epub 2013 Sep 17.
Natural products are considered an extremely valuable source for the discovery of new drugs against diverse pathologies. As yet, we have only explored a fraction of the diversity of bioactive compounds, and opportunities for discovering new natural products leading to new drugs are huge. In the present study, U2nesRELOC, a previously established cell-based imaging assay, was employed to screen a collection of extracts of microbial origin for nuclear export inhibition activity. The fluorescent signal of untreated U2nesRELOC cells localizes predominantly to the cytoplasm. Upon treatment with the nuclear export inhibitor leptomycin B, the fluorescent-tagged reporter proteins appear as speckles in the nucleus. A proprietary collection of extracts from fungi, actinomycetes, and unicellular bacteria that covers an uncommonly broad chemical space was used to interrogate this nuclear export assay system. A two-step image-based analysis allowed us to identify 12 extracts with biological activities that are not associated with previously known active metabolites. The fractionation and structural elucidation of active compounds revealed several chemical structures with nuclear export inhibition activity. Here we show that substrates of the nuclear export receptor CRM1, such as Rev, FOXO3a and NF-κB, accumulate in the nucleus in the presence of the fungal metabolite MDN-0105 with an IC50 value of 3.4 µM. Many important processes in tumor formation and progression, as well as in many viral infections, critically depend on the nucleocytoplasmic trafficking of proteins and RNA molecules. Therefore, the disruption of nuclear export is emerging as a novel therapeutic approach with enormous clinical potential. Our work highlights the potential of applying high-throughput phenotypic imaging on natural product extracts to identify novel nuclear export inhibitors.
天然产物被认为是发现针对各种病理的新药的极其宝贵的来源。迄今为止，我们仅探索了生物活性化合物多样性的一小部分，发现可导致新药的新天然产物的机会巨大。在本研究中，采用先前建立的基于细胞的成像测定法U2nesRELOC，来筛选微生物来源的提取物库，以检测其核输出抑制活性。未处理的U2nesRELOC细胞的荧光信号主要定位于细胞质。在用核输出抑制剂雷帕霉素B处理后，荧光标记的报告蛋白在细胞核中呈现为斑点。使用一个来自真菌、放线菌和单细胞细菌的提取物的专有文库，该文库覆盖了异常广阔的化学空间，来检测这个核输出测定系统。基于图像的两步分析使我们能够鉴定出12种具有生物活性的提取物，这些活性与先前已知的活性代谢物无关。活性化合物的分离和结构解析揭示了几种具有核输出抑制活性的化学结构。在这里，我们表明核输出受体CRM1的底物，如Rev、FOXO3a和NF-κB，在真菌代谢物MDN-0105存在的情况下会在细胞核中积累，IC50值为3.4μM。肿瘤形成和进展以及许多病毒感染中的许多重要过程，严重依赖于蛋白质和RNA分子的核质运输。因此，核输出的破坏正在成为一种具有巨大临床潜力的新型治疗方法。我们的工作突出了对天然产物提取物应用高通量表型成像以鉴定新型核输出抑制剂的潜力。
