McPhie Katherine A, Esposito Diego, Pettinger Jonathan, Norman Daniel, Werner Thilo, Mathieson Toby, Bush Jacob T, Rittinger Katrin
Molecular Structure of Cell Signalling Laboratory, The Francis Crick Institute 1 Midland Road London NW1 1AT UK
Crick-GSK Biomedical LinkLabs, GSK Gunnels Wood Road, Stevenage Hertfordshire SG1 2NY UK.
Chem Sci. 2025 May 12. doi: 10.1039/d5sc01540e.
The tripartite motif (TRIM) family of RING-type E3 ligases catalyses the formation of many different types of ubiquitin chains, and as such, plays important roles in diverse cellular functions, ranging from immune regulation to cancer signalling pathways. Few ligands have been discovered for TRIM E3 ligases, and these E3s are under-represented in the rapidly expanding field of induced proximity. Here we present the identification of a novel covalent ligand for the PRYSPRY substrate binding domain of TRIM25. We employ covalent fragment screening coupled with high-throughput chemistry direct-to-biology optimisation to efficiently elaborate covalent fragment hits. We demonstrate that our optimised ligand enhances the auto-ubiquitination activity of TRIM25 and engages TRIM25 in live cells. We also present the X-ray crystal structure of TRIM25 PRYSPRY in complex with this covalent ligand. Finally, we incorporate our optimised ligand into heterobifunctional proximity-inducing compounds and demonstrate the targeted ubiquitination of a neosubstrate by TRIM25.
指环型E3连接酶的三元基序（TRIM）家族催化多种不同类型泛素链的形成，因此在从免疫调节到癌症信号通路等多种细胞功能中发挥重要作用。针对TRIM E3连接酶发现的配体很少，并且这些E3在迅速发展的诱导邻近领域中所占比例不足。在此，我们展示了一种针对TRIM25的PRYSPRY底物结合结构域的新型共价配体的鉴定。我们采用共价片段筛选结合高通量化学直接到生物学的优化方法，有效地完善共价片段命中物。我们证明，我们优化后的配体增强了TRIM25的自身泛素化活性，并在活细胞中与TRIM25结合。我们还展示了与这种共价配体结合的TRIM25 PRYSPRY的X射线晶体结构。最后，我们将优化后的配体整合到异双功能邻近诱导化合物中，并证明TRIM25对新底物的靶向泛素化作用。
