USP28通过调控c-Myc和ΔNp63等致癌因子的寿命参与了肿瘤发生，并被发现是抗癌药物开发的潜在靶点。目前已经开发了大量USP28抑制剂，但它们都仍处于临床前研究阶段。此外，其中没有一个显示出令人满意的针对其最接近的同源物USP25的选择性抑制作用。本文通过高通量筛选试验，旨在发现具有新颖骨架和增强选择性抑制作用的USP28抑制剂。经过初步筛选和第二轮验证，我们发现已批准用于治疗肠易激综合征的溴酸奥替（Otilonium Bromide）能够以IC50值为6.90±0.90μM抑制USP28的活性。此外，该药物在抑制USP28超过USP25方面展示出3-4倍的选择性。根据酶动力学分析数据和氢-氘交换质谱结果，溴酸奥替能够与USP28的变构口袋结合并以可逆的非竞争性方式抑制其活性。通过以下细胞实验，我们发现该药物可能通过下调USP28的致癌底物c-Myc和/或ΔNp63来导致对人类结直肠癌细胞和肺鳞癌细胞的细胞毒性。同时，由于已发现溴酸奥替更倾向于分布在胃肠组织中，我们评估了其在与已批准用于结直肠癌治疗的雷歌替尼（Regorafenib）的联合治疗中的潜力。正如预期的那样，溴酸奥替能够显著增强结直肠癌细胞对雷歌替尼的敏感性。 版权所有 © 2023. 由Elsevier Inc.出版。

USP28 contributes to tumorigenesis through modulating the lifespan of oncogenic factors such as c-Myc and ΔNp63, and it has been identified as a potential target for anti-cancer drug development. Currently, although quite a number of USP28 inhibitors have been developed, they all are still in preclinical research stage. Besides, none of them exhibits satisfying inhibition selectivity against USP28 over its closest homologue USP25. Here in this manuscript, a high-throughput screening aiming to discover USP28 inhibitors with novel scaffold and enhanced inhibition selectivity were conducted. After the primary screening and the second round of validation, Otilonium Bromide, an approved drug for treating irritable bowel syndrome, was identified to inhibit USP28's activity with the IC50 value at 6.90 ± 0.90 μM. Besides, the drug exhibits a 3-4 folds inhibition selectivity against USP28 over USP25. According to the enzymatic kinetics analysis data and the hydrogen-deuterium exchange mass spectrometry results, Otilonium Bromide could bind to the allosteric pocket of USP28 and inhibit its activity in a reversible and non-competitive mode. The following performed cell-based assays revealed that the drug could cause cytotoxicity against human colorectal cancer cells and lung squamous carcinoma cells potentially through down-regulating USP28's oncogenic substrates c-Myc and/or ΔNp63. Meanwhile, since Otilonium Bromide has been found to preferentially distribute to gastrointestinal tissues, we then evaluated its potential in the combination treatment of colorectal cancer cells with Regorafenib, which is an approved drug for colorectal cancer therapy. As expected, Otilonium Bromide could significantly enhance the sensitivity of colorectal cancer cells to Regorafenib.Copyright © 2023. Published by Elsevier Inc.