Yang Ka, Zhao Yu, Nie Xueqing, Wu Hao, Wang Bo, Almodovar-Rivera Chelsi M, Xie Haibo, Tang Weiping
School of Pharmacy, University of Wisconsin-Madison, Madison, WI 53705, USA.
School of Pharmacy, University of Wisconsin-Madison, Madison, WI 53705, USA.
Cell Chem Biol. 2020 Jul 16;27(7):866-876.e8. doi: 10.1016/j.chembiol.2020.04.008. Epub 2020 May 14.
Proteolysis-targeting chimeras (PROTACs) is a paradigm shift for small-molecule drug discovery. However, limited E3 ubiquitin ligase ligands with cellular activity are available. In vitro binding assays involve the expression and purification of a large amount of proteins and they often yield ligands that are inactive in cell-based assays due to poor cell permeability, stability, and other reasons. Herein, we report the development of a practical and efficient cell-based target engagement assay to evaluate the binding affinity of a small library of cereblon ligands to its E3 ligase in cells. Selected cell-permeable E3 ligase ligands derived from this assay are then used to construct HDAC6 degraders with cellular protein degradation activity. Because the assay does not involve any genetic engineering, it is relatively easy to transfer from one cell type to a different one.
蛋白酶靶向嵌合体（PROTACs）是小分子药物发现领域的一次范式转变。然而，具有细胞活性的E3泛素连接酶配体数量有限。体外结合试验需要大量蛋白质的表达和纯化，而且由于细胞通透性差、稳定性不佳及其他原因，这些试验所产生的配体在基于细胞的试验中往往没有活性。在此，我们报告了一种实用且高效的基于细胞的靶点结合试验的开发，用于评估一小部分大脑神经酰胺配体文库与细胞中E3连接酶的结合亲和力。然后，从该试验中筛选出具有细胞通透性的E3连接酶配体，用于构建具有细胞内蛋白质降解活性的HDAC6降解剂。由于该试验不涉及任何基因工程，因此相对容易从一种细胞类型转移到另一种细胞类型。
