McShane Marisa P, Friedrichson Tim, Giner Angelika, Meyenhofer Felix, Barsacchi Rico, Bickle Marc, Zerial Marino
Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany.
JADO Technologies GmbH, Dresden, Germany.
J Biomol Screen. 2015 Jul;20(6):720-8. doi: 10.1177/1087057115579613. Epub 2015 Apr 2.
High-content screening of compound libraries poses various challenges in the early steps in drug discovery such as gaining insights into the mode of action of the selected compounds. Here, we addressed these challenges by integrating two biological screens through bioinformatics and computational analysis. We screened a small-molecule library enriched in amphiphilic compounds in a degranulation assay in rat basophilic leukemia 2H3 (RBL-2H3) cells. The same library was rescreened in a high-content image-based endocytosis assay in HeLa cells. This assay was previously applied to a genome-wide RNAi screen that produced quantitative multiparametric phenotypic profiles for genes that directly or indirectly affect endocytosis. By correlating the endocytic profiles of the compounds with the genome-wide siRNA profiles, we identified candidate pathways that may be inhibited by the compounds. Among these, we focused on the Akt pathway and validated its inhibition in HeLa and RBL-2H3 cells. We further showed that the compounds inhibited the translocation of the Akt-PH domain to the plasma membrane. The approach performed here can be used to integrate chemical and functional genomics screens for investigating the mechanism of action of compounds.
在药物发现的早期阶段，对化合物库进行高内涵筛选面临着各种挑战，比如深入了解所选化合物的作用模式。在此，我们通过生物信息学和计算分析整合两种生物学筛选方法来应对这些挑战。我们在大鼠嗜碱性白血病2H3（RBL - 2H3）细胞的脱颗粒试验中筛选了一个富含两亲性化合物的小分子库。同一文库在HeLa细胞的基于高内涵图像的内吞作用试验中进行了重新筛选。该试验先前已应用于全基因组RNA干扰筛选，该筛选为直接或间接影响内吞作用的基因生成了定量多参数表型图谱。通过将化合物的内吞图谱与全基因组siRNA图谱相关联，我们确定了可能被这些化合物抑制的候选通路。其中，我们重点研究了Akt通路，并在HeLa和RBL - 2H3细胞中验证了其抑制作用。我们进一步表明，这些化合物抑制了Akt - PH结构域向质膜的转位。这里所采用的方法可用于整合化学和功能基因组学筛选，以研究化合物的作用机制。
