Suppr超能文献

基于片段的共价配体筛选可快速发现 RBR E3 泛素连接酶 HOIP 的抑制剂。

Fragment-Based Covalent Ligand Screening Enables Rapid Discovery of Inhibitors for the RBR E3 Ubiquitin Ligase HOIP.

机构信息

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.

出版信息

J Am Chem Soc. 2019 Feb 13;141(6):2703-2712. doi: 10.1021/jacs.8b13193. Epub 2019 Feb 4.

Abstract

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.

摘要

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。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/d1f8/6383986/e7111127c201/ja-2018-13193s_0001.jpg

相似文献

1
Fragment-Based Covalent Ligand Screening Enables Rapid Discovery of Inhibitors for the RBR E3 Ubiquitin Ligase HOIP.
J Am Chem Soc. 2019 Feb 13;141(6):2703-2712. doi: 10.1021/jacs.8b13193. Epub 2019 Feb 4.
3
Single-Domain Antibodies as Crystallization Chaperones to Enable Structure-Based Inhibitor Development for RBR E3 Ubiquitin Ligases.
Cell Chem Biol. 2020 Jan 16;27(1):83-93.e9. doi: 10.1016/j.chembiol.2019.11.007. Epub 2019 Dec 5.
4
Structure of a HOIP/E2~ubiquitin complex reveals RBR E3 ligase mechanism and regulation.
Nature. 2016 Jan 28;529(7587):546-50. doi: 10.1038/nature16511. Epub 2016 Jan 20.
5
The HOIL-1L ligase modulates immune signalling and cell death via monoubiquitination of LUBAC.
Nat Cell Biol. 2020 Jun;22(6):663-673. doi: 10.1038/s41556-020-0517-9. Epub 2020 May 11.
7
LUBAC synthesizes linear ubiquitin chains via a thioester intermediate.
EMBO Rep. 2012 Sep;13(9):840-6. doi: 10.1038/embor.2012.105. Epub 2012 Jul 13.
8
Linear ubiquitin chains: enzymes, mechanisms and biology.
Open Biol. 2017 Apr;7(4). doi: 10.1098/rsob.170026.
10
Mechanistic insights into the subversion of the linear ubiquitin chain assembly complex by the E3 ligase IpaH1.4 of .
Proc Natl Acad Sci U S A. 2022 Mar 22;119(12):e2116776119. doi: 10.1073/pnas.2116776119. Epub 2022 Mar 16.

引用本文的文献

1
COOKIE-Pro: Covalent Inhibitor Binding Kinetics Profiling on the Proteome Scale.
bioRxiv. 2025 Jun 22:2025.06.19.660637. doi: 10.1101/2025.06.19.660637.
2
Spatiotemporal Regulation of STING Activity by Linear Ubiquitination Governs Antiviral Immunity.
Adv Sci (Weinh). 2025 Jul;12(28):e2417660. doi: 10.1002/advs.202417660. Epub 2025 Jun 19.
3
Computational Design of Lysine Targeting Covalent Binders Using Rosetta.
J Chem Inf Model. 2025 May 29. doi: 10.1021/acs.jcim.5c00212.
4
Robust proteome profiling of cysteine-reactive fragments using label-free chemoproteomics.
Nat Commun. 2025 Jan 2;16(1):73. doi: 10.1038/s41467-024-55057-5.
5
Evaluation of a Covalent Library of Diverse Warheads (CovLib) Binding to JNK3, USP7, or p53.
Drug Des Devel Ther. 2024 Jul 1;18:2653-2679. doi: 10.2147/DDDT.S466829. eCollection 2024.
6
Breaking Bad Proteins-Discovery Approaches and the Road to Clinic for Degraders.
Cells. 2024 Mar 26;13(7):578. doi: 10.3390/cells13070578.
7
The cross talk of ubiquitination and chemotherapy tolerance in colorectal cancer.
J Cancer Res Clin Oncol. 2024 Mar 23;150(3):154. doi: 10.1007/s00432-024-05659-9.
8
Mechanisms underlying linear ubiquitination and implications in tumorigenesis and drug discovery.
Cell Commun Signal. 2023 Nov 28;21(1):340. doi: 10.1186/s12964-023-01239-5.
9
The mechanism of linear ubiquitination in regulating cell death and correlative diseases.
Cell Death Dis. 2023 Oct 10;14(10):659. doi: 10.1038/s41419-023-06183-3.
10
Repressive Control of Keratinocyte Cytoplasmic Inflammatory Signaling.
Int J Mol Sci. 2023 Jul 26;24(15):11943. doi: 10.3390/ijms241511943.

本文引用的文献

1
The MALDI-TOF E2/E3 Ligase Assay as Universal Tool for Drug Discovery in the Ubiquitin Pathway.
Cell Chem Biol. 2018 Sep 20;25(9):1117-1127.e4. doi: 10.1016/j.chembiol.2018.06.004. Epub 2018 Jul 12.
3
Biophysical and biological evaluation of optimized stapled peptide inhibitors of the linear ubiquitin chain assembly complex (LUBAC).
Bioorg Med Chem. 2018 Mar 15;26(6):1179-1188. doi: 10.1016/j.bmc.2017.11.047. Epub 2017 Dec 5.
4
The Met1-Linked Ubiquitin Machinery: Emerging Themes of (De)regulation.
Mol Cell. 2017 Oct 19;68(2):265-280. doi: 10.1016/j.molcel.2017.09.001.
5
Linear ubiquitin chains: enzymes, mechanisms and biology.
Open Biol. 2017 Apr;7(4). doi: 10.1098/rsob.170026.
6
Ligand and Target Discovery by Fragment-Based Screening in Human Cells.
Cell. 2017 Jan 26;168(3):527-541.e29. doi: 10.1016/j.cell.2016.12.029. Epub 2017 Jan 19.
7
A Perspective on the Kinetics of Covalent and Irreversible Inhibition.
SLAS Discov. 2017 Jan;22(1):3-20. doi: 10.1177/1087057116671509. Epub 2016 Oct 5.
8
A Modular Probe Strategy for Drug Localization, Target Identification and Target Occupancy Measurement on Single Cell Level.
ACS Chem Biol. 2016 Sep 16;11(9):2541-50. doi: 10.1021/acschembio.6b00346. Epub 2016 Jul 20.
9
Proteome-wide covalent ligand discovery in native biological systems.
Nature. 2016 Jun 23;534(7608):570-4. doi: 10.1038/nature18002. Epub 2016 Jun 15.
10
Drugging the undruggables: exploring the ubiquitin system for drug development.
Cell Res. 2016 Apr;26(4):484-98. doi: 10.1038/cr.2016.31. Epub 2016 Mar 22.

文献AI研究员

20分钟写一篇综述,助力文献阅读效率提升50倍。

立即体验

用中文搜PubMed

大模型驱动的PubMed中文搜索引擎

马上搜索

文档翻译

学术文献翻译模型,支持多种主流文档格式。

立即体验