A resurging interest in targeted covalent inhibitors (TCIs) focus on compounds capable of irreversibly reacting with nucleophilic amino acids in a druggable target. p97 is an emerging protein target for cancer therapy, viral infections and neurodegenerative diseases. Extensive efforts were devoted to the development of p97 inhibitors. The most promising inhibitor of p97 was in phase 1 clinical trials, but failed due to the off-target-induced toxicity, suggesting the selective inhibitors of p97 are highly needed. We report herein a new type of TCIs (i.e., FL-18) that showed proteome-wide selectivity towards p97. Equipped with a Michael acceptor and a basic imidazole, FL-18 showed potent inhibition towards U87MG tumor cells, and in proteome-wide profiling, selectively modified endogenous p97 as confirmed by in situ fluorescence scanning, label-free quantitative proteomics and functional validations. FL-18 selectively modified cysteine residues located within the D2 ATP site of p97. This covalent labeling of cysteine residue in p97 was verified by LC‒MS/MS-based site-mapping and site-directed mutagenesis. Further structure–activity relationship (SAR) studies with FL-18 analogs were established. Collectively, FL-18 is the first known small-molecule TCI capable of covalent engagement of p97 with proteome-wide selectivity, thus providing a promising scaffold for cancer therapy.

对靶向共价抑制剂 (TCI) 的重新关注集中在能够与可成​​药靶标中的亲核氨基酸发生不可逆反应的化合物上。p97 是癌症治疗、病毒感染和神经退行性疾病的新兴蛋白质靶点。广泛的努力致力于p97抑制剂的开发。最有希望的 p97 抑制剂处于 1 期临床试验中，但由于脱靶诱导的毒性而失败，这表明 p97 的选择性抑制剂非常需要。我们在此报告了一种新型的 TCI（即FL-18），它显示出对 p97 的全蛋白质组选择性。配备 Michael 受体和碱性咪唑，FL-18 显示出对 U87MG 肿瘤细胞的有效抑制作用，在蛋白质组分析中，选择性修饰的内源性 p97 证实了这一点原位荧光扫描、无标记定量蛋白质组学和功能验证。FL-18 选择性修饰位于 p97 的 D2 ATP 位点内的半胱氨酸残基。p97 中半胱氨酸残基的共价标记通过基于 LC-MS/MS 的位点作图和定点诱变得到验证。建立了与 FL-18 类似物的进一步构效关系 (SAR) 研究。总的来说，FL-18 是第一个已知的能够与 p97 共价结合并具有全蛋白质组选择性的小分子 TCI，从而为癌症治疗提供了有希望的支架。