Fragment-Based Covalent Ligand Screening Enables Rapid Discovery of Inhibitors for the RBR E3 Ubiquitin Ligase HOIP
Fragment-Based Covalent Ligand Screening Enables Rapid Discovery of Inhibitors for the RBR E3 Ubiquitin Ligase HOIP
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DOI：
10.1021/jacs.8b13193
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发表时间：
2019-02-13
影响因子：
15
通讯作者：
Rittinger, Katrin
中科院分区：
文献类型：
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作者：
Johansson, Henrik;Tsai, Yi-Chun Isabella;Rittinger, Katrin
用多聚泛素链修饰蛋白质是控制细胞行为的关键调节机制，并且泛素系统的改变与许多疾病有关。线性（M1连接的）多聚泛素链在介导免疫和炎症反应以及凋亡性细胞死亡的几种细胞信号传导途径中发挥关键作用。这些链由线性泛素链组装复合物（LUBAC）形成，LUBAC是一种多蛋白E3连接酶，由3个亚基HOIP、HOIL-1 L和SHARPIN组成。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的活性位点半胱氨酸的抑制剂的发现。我们报告了一个不同的亲电片段库的合成，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，使第一个基于结构的开发RBR E3连接酶的共价抑制剂。此外，使用基于细胞的测定和化学蛋白质组学，我们证明了这些化合物有效地渗透哺乳动物细胞，以标记和抑制HOIP和NF-κ B活化，使它们适合用于开发选择性探针以研究LUBAC生物学。我们的研究结果说明了基于片段的共价配体筛选的力量，以发现具有挑战性的目标，这有望成为一种通用的方法，用于开发硫酯形成E3泛素连接酶的细胞渗透性抑制剂的先导化合物。
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-kappa B 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.