Centre for Medicines Discovery, Nuffield Department of Medicine, University of Oxford, Old Road Campus, Roosevelt Drive, Oxford OX3 7FZ, U.K.
Target Discovery Institute, Nuffield Department of Medicine, University of Oxford, Old Road Campus, Roosevelt Drive, Oxford OX3 7FZ, U.K.
J Med Chem. 2024 May 9;67(9):7245-7259. doi: 10.1021/acs.jmedchem.4c00072. Epub 2024 Apr 18.
Cofactor mimicry represents an attractive strategy for the development of enzyme inhibitors but can lead to off-target effects due to the evolutionary conservation of binding sites across the proteome. Here, we uncover the ADP-ribose (ADPr) hydrolase NUDT5 as an unexpected, noncovalent, off-target of clinical BTK inhibitors. Using a combination of biochemical, biophysical, and intact cell NanoBRET assays as well as X-ray crystallography, we confirm catalytic inhibition and cellular target engagement of NUDT5 and reveal an unusual binding mode that is independent of the reactive acrylamide warhead. Further investigation of the prototypical BTK inhibitor ibrutinib also revealed potent inhibition of the largely unstudied NUDIX hydrolase family member NUDT14. By exploring structure-activity relationships (SARs) around the core scaffold, we identify a potent, noncovalent, and cell-active dual NUDT5/14 inhibitor. Cocrystallization experiments yielded new insights into the NUDT14 hydrolase active site architecture and inhibitor binding, thus providing a basis for future chemical probe design.
辅因子模拟代表了开发酶抑制剂的一种有吸引力的策略，但由于结合位点在整个蛋白质组中的进化保守性，可能会导致非靶向效应。在这里，我们发现 ADP-核糖 (ADPr) 水解酶 NUDT5 是临床 BTK 抑制剂的一个意外的、非共价的、非靶标。我们使用生化、生物物理和完整细胞 NanoBRET 测定以及 X 射线晶体学的组合，证实了 NUDT5 的催化抑制和细胞靶标结合，并揭示了一种不依赖于反应性丙烯酰胺弹头的不寻常结合模式。对典型的 BTK 抑制剂伊布替尼的进一步研究还揭示了对研究甚少的 NUDIX 水解酶家族成员 NUDT14 的强烈抑制作用。通过探索核心支架周围的结构-活性关系 (SAR)，我们确定了一种有效的、非共价的、具有细胞活性的双重 NUDT5/14 抑制剂。共结晶实验为 NUDT14 水解酶活性位点结构和抑制剂结合提供了新的见解，从而为未来的化学探针设计提供了基础。
