Crick-GSK Biomedical LinkLabs , GlaxoSmithKline , Gunnels Wood Road , Stevenage SG1 2NY , United Kingdom.
Molecular Structure of Cell Signalling Laboratory , The Francis Crick Institute , 1 Midland Road , London NW1 1AT , United Kingdom.
J Am Chem Soc. 2019 Feb 13;141(6):2703-2712. doi: 10.1021/jacs.8b13193. Epub 2019 Feb 4.
Modification of proteins with polyubiquitin chains is a key regulatory mechanism to control cellular behavior and alterations in the ubiquitin system are linked to many diseases. Linear (M1-linked) polyubiquitin chains play pivotal roles in several cellular signaling pathways mediating immune and inflammatory responses and apoptotic cell death. These chains are formed by the linear ubiquitin chain assembly complex (LUBAC), a multiprotein E3 ligase that consists of 3 subunits, HOIP, HOIL-1L, and SHARPIN. Herein, we describe the discovery of inhibitors targeting the active site cysteine of the catalytic subunit HOIP using fragment-based covalent ligand screening. We report the synthesis of a diverse library of electrophilic fragments and demonstrate an integrated use of protein LC-MS, biochemical ubiquitination assays, chemical synthesis, and protein crystallography to enable the first structure-based development of covalent inhibitors for an RBR E3 ligase. Furthermore, using cell-based assays and chemoproteomics, we demonstrate that these compounds effectively penetrate mammalian cells to label and inhibit HOIP and NF-κB activation, making them suitable hits for the development of selective probes to study LUBAC biology. Our results illustrate the power of fragment-based covalent ligand screening to discover lead compounds for challenging targets, which holds promise to be a general approach for the development of cell-permeable inhibitors of thioester-forming E3 ubiquitin ligases.
多聚泛素链修饰蛋白是控制细胞行为的关键调节机制,泛素系统的改变与许多疾病有关。线性(M1 连接)多聚泛素链在介导免疫和炎症反应以及细胞凋亡的几种细胞信号通路中发挥关键作用。这些链由线性泛素链组装复合物(LUBAC)形成,LUBAC 是一种由 3 个亚基组成的多蛋白 E3 连接酶,包括 HOIP、HOIL-1L 和 SHARPIN。在此,我们描述了使用基于片段的共价配体筛选靶向催化亚基 HOIP 活性位点半胱氨酸的抑制剂的发现。我们报告了一个多样化的亲电子片段文库的合成,并展示了蛋白质 LC-MS、生化泛素化测定、化学合成和蛋白质晶体学的综合使用,从而首次为 RBR E3 连接酶开发基于结构的共价抑制剂。此外,通过基于细胞的测定和化学蛋白质组学,我们证明这些化合物能够有效地穿透哺乳动物细胞标记和抑制 HOIP 和 NF-κB 激活,使其成为研究 LUBAC 生物学的选择性探针开发的合适候选物。我们的结果说明了基于片段的共价配体筛选发现挑战性靶标先导化合物的强大功能,这有望成为开发透细胞膜硫酯形成 E3 泛素连接酶抑制剂的一般方法。