Abstract：
Proteolysis targeting chimeras (PROTACs) are new chemical modalities that degrade proteins of interest, including established kinase targets and emerging RNA-binding proteins (RBPs). Whereas diverse sets of biochemical, biophysical and cellular assays are available for the evaluation and optimizations of PROTACs in understanding the involved ubiquitin-proteasome-mediated degradation mechanism and the structure-degradation relationship, a phenotypic method profiling the cellular morphological changes is rarely used. In this study, first, we reported the only examples of PROTACs degrading the mRNA-binding protein YTHDF2 via screening of multikinase PROTACs. Second, we reported the profiling of cellular morphological changes of the dual kinase- and RBP-targeting PROTACs using the unbiased cell painting assay (CPA). The CPA analysis revealed the high biosimilarity with the established aurora kinase cluster and annotated aurora kinase inhibitors, which reflected the association between YTHDF2 and the aurora kinase signaling network. Broadly, the results demonstrated that the cell painting assay can be a straightforward and powerful approach to evaluate PROTACs. Complementary to the existing biochemical, biophysical and cellular assays, CPA provided a new perspective in characterizing PROTACs at the cellular morphology.
摘要：
蛋白水解靶向嵌合体（PROTACs）是降解感兴趣的蛋白质的新化学模式，包括已建立的激酶靶标和新兴的RNA结合蛋白（RBP）。而不同的生化组，生物物理和细胞分析可用于评估和优化PROTACs，以了解所涉及的泛素-蛋白酶体介导的降解机制和结构-降解关系，很少使用表型方法分析细胞形态变化。在这项研究中,首先,我们报道了通过筛选多激酶PROTACs降解mRNA结合蛋白YTHDF2的唯一例子.第二,我们报道了使用无偏倚细胞涂漆试验(CPA)对双重激酶和RBP靶向PROTACs细胞形态变化的分析.CPA分析显示与已建立的极光激酶簇和注释的极光激酶抑制剂具有高度的生物相似性。这反映了YTHDF2与极光激酶信号网络之间的关联。广义上,结果表明，细胞涂漆测定法可以是评估PROTACs的简单而有效的方法。补充现有的生化，生物物理和细胞测定，CPA为表征细胞形态的PROTACs提供了新的视角。
