Ubiquitin-specific protease 30 (USP30) is a deubiquitinating enzyme (DUB) localized at the mitochondrial outer membrane and involved in PINK1/Parkin-mediated mitophagy, pexophagy, BAX/BAK-dependent apoptosis, and IKKβ-USP30-ACLY-regulated lipogenesis/tumorigenesis. A USP30 inhibitor, MTX652, has recently entered clinical trials as a potential treatment for mitochondrial dysfunction. Small molecule activity-based probes (ABPs) for DUBs have recently emerged as powerful tools for in-cell inhibitor screening and DUB activity analysis, and here, we report the first small molecule ABPs (IMP-2587 and IMP-2586) which can profile USP30 activity in cells. Target engagement studies demonstrate that IMP-2587 and IMP-2586 engage active USP30 at nanomolar concentration after only 10 min incubation time in intact cells, dependent on the presence of the USP30 catalytic cysteine. Interestingly, proteomics analyses revealed that DESI1 and DESI2, small ubiquitin-related modifier (SUMO) proteases, can also be engaged by these probes, further suggesting a novel approach to develop DESI ABPs.

中文翻译：

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发现去泛素化酶 USP30 的有效且选择性的基于活性的探针 (ABP)

泛素特异性蛋白酶 30 (USP30) 是一种位于线粒体外膜的去泛素化酶 (DUB)，参与 PINK1/Parkin 介导的线粒体自噬、pexophagy、BAX/BAK 依赖性细胞凋亡和 IKKβ-USP30-ACLY 调节的脂肪生成/肿瘤发生。USP30 抑制剂MTX652最近已进入临床试验，作为线粒体功能障碍的潜在治疗方法。基于小分子活性的 DUB 探针 (ABP) 最近已成为细胞内抑制剂筛选和 DUB 活性分析的强大工具，在这里，我们报告了第一个可以分析的小分子 ABP（IMP-2587和IMP-2586）细胞中的 USP30 活性。靶标接合研究表明，IMP-2587和IMP-2586在完整细胞中仅孵育 10 分钟后即可以纳摩尔浓度接合活性 USP30，具体取决于 USP30 催化半胱氨酸的存在。有趣的是，蛋白质组学分析表明，DESI1 和 DESI2（小型泛素相关修饰剂 (SUMO)）蛋白酶也可以被这些探针参与，进一步提出了一种开发 DESI ABP 的新方法。