Due to their role in many diseases, enzymes of the ubiquitin system have recently become interesting drug targets. Despite efforts, primary screenings of compound libraries targeting E2 enzymes and E3 ligases have been strongly limited by the lack of robust and fast high-throughput assays. Here we report a label-free high-throughput screening assay for ubiquitin E2 conjugating enzymes and E3 ligases based on MALDI-TOF mass spectrometry. The MALDI-TOF E2/E3 assay allows testing E2 enzymes and E3 ligases for their ubiquitin transfer activity, identifying E2/E3 active pairs, inhibitor potency and specificity and screening compound librariesin vitrowithout chemical or fluorescent probes. We demonstrate that the MALDI-TOF E2/E3 assay is a universal tool for drug discovery screening in the ubiquitin pathway as it is suitable for working with all E3 ligase families and requires a reduced amount of reagents, compared with standard biochemical assays.

中文翻译：

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MALDI-TOF E2/E3 连接酶检测作为泛素途径药物发现的通用工具

由于它们在许多疾病中的作用，泛素系统的酶最近已成为有趣的药物靶标。尽管做出了努力，但针对 E2 酶和 E3 连接酶的化合物文库的初步筛选由于缺乏稳健和快速的高通量测定而受到严重限制。在这里，我们报告了一种基于 MALDI-TOF 质谱法的泛素 E2 结合酶和 E3 连接酶的无标记高通量筛选试验。MALDI-TOF E2/E3 测定允许测试 E2 酶和 E3 连接酶的泛素转移活性，鉴定 E2/E3 活性对、抑制剂效力和特异性，并在体外筛选化合物库，无需化学或荧光探针。