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.

靶向蛋白水解的嵌合体（PROTAC）是小分子药物发现的范例转变。但是，有限的具有细胞活性的E3泛素连接酶配体是可用的。体外结合测定涉及大量蛋白质的表达和纯化，由于细胞通透性，稳定性和其他原因，它们通常会产生在基于细胞的测定中失活的配体。在本文中，我们报告了一种实用，有效的基于细胞的靶标结合分析技术的开发，以评估小脑神经配体文库与其在细胞中的E3连接酶的结合亲和力。然后，从该测定中选择的细胞可渗透的E3连接酶配体用于构建具有细胞蛋白降解活性的HDAC6降解剂。因为该测定法不涉及任何基因工程，所以从一种细胞类型转移到另一种细胞类型相对容易。